CN111527198A - Cell population comprising adherent stem cells, method for producing same, and pharmaceutical composition - Google Patents

Cell population comprising adherent stem cells, method for producing same, and pharmaceutical composition Download PDF

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CN111527198A
CN111527198A CN201880084442.5A CN201880084442A CN111527198A CN 111527198 A CN111527198 A CN 111527198A CN 201880084442 A CN201880084442 A CN 201880084442A CN 111527198 A CN111527198 A CN 111527198A
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stem cells
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梅田伸好
稻生溪太
小林千穗
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Kaneka Corp
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/115—Platelets, megakaryocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease

Abstract

The present invention addresses the problem of providing a cell population that contains safe adherent stem cells that retain a normal karyotype, a method for producing the same, and a pharmaceutical composition that contains the cell population. According to the present invention, there can be provided a method for producing a cell population comprising adherent stem cells, the method comprising: obtaining a cell population in which the ratio of adherent stem cells positive for KCNAB1 is 85% or more.

Description

Cell population comprising adherent stem cells, method for producing same, and pharmaceutical composition
Technical Field
The present invention relates to a cell population of adherent stem cells such as mesenchymal stem cells. The present invention relates to a method for producing the cell population and a pharmaceutical composition containing the cell population. The present invention also relates to a method for monitoring karyotypic abnormality (karyotypicabnormality) of adherent stem cells using, as an indicator, the ratio of adherent stem cells expressing a specific marker in a cell population, a method for evaluating a donor and/or a biological sample collected from the donor, and a method for judging and/or predicting enzyme treatment conditions.
Background
Adherent stem cells such as Mesenchymal stem cells, also called Mesenchymal stromal cells (mesenchymeal stromal cells), are adult stem cells reported to be present in bone marrow, adipose tissue, dental pulp, and the like, and have recently been found to be present in fetal appendages such as placenta, umbilical cord, fetal membrane, and the like. Furthermore, the adherent stem cells have immunosuppressive ability, and have been put to practical use for acute Graft Versus Host Disease (GVHD), crohn's disease as inflammatory bowel disease, and the like.
In addition, in recent years, there has been a demand for providing safe adherent stem cells that can be sufficiently used for cell therapy applications, and for example, non-patent document 1 discloses a criterion for karyotyping of mesenchymal stem cells used for cell therapy. Non-patent document 2 describes the frequency of occurrence of karyotypic abnormalities in mesenchymal stem cells derived from bone marrow of a plurality of donors and the correlation between the number of passages and the frequency of occurrence of karyotypic abnormalities.
Documents of the prior art
Non-patent document
Non-patent document 1: lisbeth Barkhole et al, Risk of genetic in genetic structural cellular peptides-Bridging genetic organisms and genetic viruses, Cytotherapy,2013,15,753-
Non-patent document 2: brian G.Stultz et al, Chromosomal Stability of Mensenchyl Stromcells During In Vitro Culture, Cytotherapy,2016,18(3),336-
Disclosure of Invention
Problems to be solved by the invention
The present inventors have conducted studies to provide safe adherent stem cells that can be sufficiently used for cell therapy, and as a result, have confirmed that karyotypic abnormalities occur at a high frequency during the culture of adherent stem cells, and that karyotypic abnormalities accumulate with passage. Since the above-mentioned problems lead to a risk of tumorigenicity (tumorigenicity), even an adherent stem cell population containing a small amount of adherent stem cells for which karyotype abnormality is recognized can be discarded in its entirety. Further, the karyotype analysis method for analyzing the chromosome structure one by one to evaluate the frequency of karyotype abnormalities requires a lot of time and labor for analysis, and is also a cause of failure to ensure quality quickly. Therefore, the present inventors have found that in order to solve the above problems, it is necessary to prepare a cell population free from karyotypic abnormality and to monitor the occurrence of karyotypic abnormality over time.
To solve this problem, non-patent document 1 has been studied. Non-patent document 1 describes a criterion for determining the frequency of structural abnormalities of chromosomes in mesenchymal stem cells used for cell therapy. Further, non-patent document 1 suggests that setting the culture conditions to a level that minimizes the growth rate and the number of doublings of cells is relevant to obtaining a cell population that does not contain karyotypic abnormalities. However, the present inventors confirmed that karyotypic abnormalities occur at a high frequency even when the proliferation rate and the number of multiplication are reduced. Non-patent document 1 does not describe a method for monitoring the presence or absence of a nuclear abnormality.
Non-patent document 2 describes the correlation between the occurrence frequency and the number of passages of karyotypic abnormalities in mesenchymal stem cells derived from bone marrow of a plurality of donors and the occurrence frequency of karyotypic abnormalities. Unlike non-patent document 1, it was confirmed that the frequency of karyotypic abnormalities tended to decrease as the number of passages increased and the number of doublings increased. Thus, the relationship between the number of cell doublings and karyotypic abnormalities tends to vary according to the literature, and the mechanism of occurrence of karyotypic abnormalities is not sufficiently elucidated. In addition, it is mentioned in non-patent document 2 that karyotyping of mesenchymal stem cells obtained at each passage is useful for monitoring the chromosomal stability of mesenchymal stem cells at the time of expansion culture. However, in the karyotyping method, after mesenchymal stem cells were obtained, chromosomes were extracted from 17 to 144 cells subjected to karyotyping, and the structures of the chromosomes were observed one by the sky (spectral karyotyping) method. The above analysis requires a lot of time and labor, is difficult to monitor the occurrence of karyotype abnormalities over time, and cannot quickly evaluate the presence or absence of karyotype abnormalities. Furthermore, there is no description of a method for preparing a cell population not containing karyotypic abnormalities.
In view of the above problems, an object of the present invention is to provide a method for obtaining a cell population including adherent stem cells that retain a normal karyotype, and for rapidly evaluating the presence or absence of a karyotypic abnormality by monitoring the occurrence of a karyotypic abnormality in the cell population including adherent stem cells over a long period of time.
Means for solving the problems
As a result of intensive studies to solve the above problems, the present inventors have found that a cell population containing adherent stem cells that retain a normal karyotype can be obtained when culturing is performed under conditions in which the ratio of adherent stem cells that are positive for KCNAB1 in the cell population containing adherent stem cells is maintained at a given value or more. The present inventors have also found that the presence or absence of karyotypic abnormality in adherent stem cells can be monitored by using, as an indicator, a ratio of adherent stem cells positive for KCNAB1 in a cell population containing adherent stem cells of a given value or more. In addition, from the viewpoint of efficiently obtaining adherent stem cells that retain a normal karyotype, the quality of the donor and/or the biological sample collected from the donor can be rapidly evaluated, and further, the optimal enzyme treatment conditions for the biological sample collected from the donor can be determined and/or predicted. The present invention has been completed based on these findings.
That is, the present specification provides the following inventions.
(1) A method of making a population of cells comprising adherent stem cells, the method comprising:
obtaining a cell population in which the rate of adherent stem cells positive for KCNAB1 is 85% or more.
(2) A cell population comprising adherent stem cells, wherein,
the cell population has a ratio of adherent stem cells positive for KCNAB1 of 85% or more.
(3) The cell population according to (2), wherein the relative expression amount of the KCNAB1 gene in the cell population with respect to the expression amount of the SDHA gene is 0.05 or more.
(4) The cell population according to (2) or (3), wherein the relative expression amount of the SULT1E1 gene relative to the expression amount of the SDHA gene in the cell population is 0.1 or more.
(5) The cell population according to any one of (2) to (4), wherein the relative expression level of MN1 gene with respect to the expression level of SDHA gene in the cell population is 0.7 or more.
(6) The cell population according to any one of (2) to (5), wherein the relative expression level of the RARES 2 gene of the cell population with respect to the expression level of the SDHA gene is 0.4 or less.
(7) The cell population according to any one of (2) to (6), wherein the adherent stem cells are derived from a fetal appendage.
(8) A pharmaceutical composition comprising the cell population of any one of (2) to (7), and a pharmaceutically acceptable vehicle.
(9) A pharmaceutical composition comprising the cell population according to any one of (2) to (7) and another administrable cell.
(10) The pharmaceutical composition according to (8) or (9), wherein the 1-time dose of the adherent stem cells for human is 1012Less than one/kg body weight.
(11) The pharmaceutical composition according to any one of (8) to (10), wherein the pharmaceutical composition is an injectable preparation.
(12) The pharmaceutical composition according to any one of (8) to (10), wherein the pharmaceutical composition is a preparation for transplantation.
(13) The pharmaceutical composition according to (12), wherein the preparation for transplantation is a cell pellet or a sheet-like structure.
(14) The pharmaceutical composition according to any one of (8) to (13), which is selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, nervous system diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, connective tissue diseases, stroke, cerebral infarction, intracerebral hematoma, cerebral vascular paralysis (cerebravicular paralysis), liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides, scleroderma, diseases caused by degeneration and/or inflammation of connective tissue such as cartilage, articular cartilage defects, meniscus injury, osteochondritis dissecans, aseptic necrosis, knee osteoarthritis (knee osteoarthritis), inflammatory arthritis, rheumatoid arthritis, and the like, A therapeutic agent for diseases in ocular diseases, angiogenesis-related diseases, ischemic heart diseases, coronary heart diseases, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart diseases, wounds, epithelial injuries, fibrosis, pulmonary diseases, and cancers.
(15) A method of monitoring adherent stem cells for karyotypic abnormalities, the method comprising:
the method comprises the steps of measuring the ratio of adherent stem cells positive to KCNAB1 in a cell population containing the adherent stem cells, and monitoring the karyotype abnormality of the adherent stem cells by using the ratio of the adherent stem cells positive to KCNAB1 in the cell population of more than 85% as an index.
(16) A method of evaluating a donor and/or a biological sample collected from a donor, the method comprising:
a cell population containing adherent stem cells is collected from a donor, the ratio of adherent stem cells positive for KCNAB1 is measured, and the ratio of adherent stem cells positive for KCNAB1 in the cell population is evaluated as an index of 85% or more.
(17) A method of determining and/or predicting optimal enzyme treatment conditions for a biological sample, the method comprising:
the percentage of adherent stem cells positive for KCNAB1 in a cell population obtained by subjecting a biological sample collected from a donor to an enzyme treatment was measured, and the evaluation was performed using as an index the percentage of adherent stem cells positive for KCNAB1 in the cell population of 85% or more.
(21) A method of treating a disease, the method comprising:
administering the cell population of any one of (2) to (7) to a patient or subject in need of treatment.
(22) The method of (21), wherein the 1 dose of adherent stem cells for human is 1 × 1012Less than one/kg body weight.
(23) The method according to (21) or (22), which is an injectable preparation.
(24) The method according to (21) or (22), which is a preparation for transplantation.
(25) The pharmaceutical composition of (24), wherein the preparation for transplantation is a cell pellet or a sheet-like structure.
(26) The method according to any one of (21) to (25), wherein the disease is selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, nervous system diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, connective tissue diseases, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides, scleroderma, diseases caused by degeneration and/or inflammation of connective tissue such as cartilage, articular cartilage defects, meniscus injury, osteochondritis dissecans, aseptic necrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, ocular diseases, and the like, Angiogenesis-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart disease, trauma, epithelial injury, fibrosis, pulmonary disease, and cancer.
(31) Use of the cell population according to any one of (2) to (6) for the production of a pharmaceutical composition.
(32) The use of (31), wherein the pharmaceutical composition is 1 dose of 1 × 10 for human adherent stem cells12A pharmaceutical composition for treating or preventing obesity per kg body weight.
(33) The use according to (31) or (32), wherein the pharmaceutical composition is an injectable preparation.
(34) The use according to (31) or (32), wherein the pharmaceutical composition is a preparation for transplantation.
(35) The pharmaceutical composition of (34), wherein the preparation for transplantation is a cell pellet or a sheet-like structure.
(36) The use according to any one of (31) to (35), wherein the pharmaceutical composition is selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, nervous system diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, connective tissue diseases, cerebral stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides, scleroderma, diseases caused by degeneration and/or inflammation of connective tissues such as cartilage, articular cartilage defects, meniscus injury, osteochondritis dissecans, aseptic necrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, A therapeutic agent for diseases in eye diseases, angiogenesis-related diseases, ischemic heart diseases, coronary heart diseases, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart diseases, wounds, epithelial injuries, fibrosis, lung diseases, and cancer.
(41) The cell population according to any one of (2) to (6), which is used for the treatment of a disease.
(42) The cell population of (41), wherein 1 dose of the adherent stem cells for human is 1 × 1012Less than one/kg body weight.
(43) The cell population according to (41) or (42), which is an injectable preparation.
(44) The cell population according to (41) or (42), which is a preparation for transplantation.
(45) The pharmaceutical composition of (44), wherein the preparation for transplantation is a cell pellet or a sheet-like structure.
(46) The cell population according to any one of (41) to (45), wherein the disease is selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, nervous system diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, connective tissue diseases, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides, scleroderma, diseases caused by degeneration and/or inflammation of connective tissue such as cartilage, articular cartilage defects, meniscus injury, osteochondritis dissecans, aseptic necrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, ocular diseases, and the like, Angiogenesis-related diseases, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart disease, trauma, epithelial injury, fibrosis, pulmonary disease, and cancer.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, a cell population comprising adherent stem cells that maintain a normal karyotype can be obtained. Further, the present invention provides a method for rapidly evaluating the presence or absence of a karyotypic abnormality by monitoring the occurrence of a karyotypic abnormality in a cell population including adherent stem cells over time. In addition, according to the present invention, the positive rate of a given antigen can be used as an indicator of the formation of a cell population containing safe adherent stem cells that maintain a normal karyotype. Further, according to the present invention, a safe cell preparation (pharmaceutical composition) having high chromosome stability and suitable for clinical use can be produced.
Drawings
Figure 1 shows the results of flow cytometry analysis of the ratio of amnion adherent stem cells obtained from fetal appendages of 5 pregnant women to cells positive for KCNAB 1.
Detailed Description
The following description will specifically describe embodiments of the present invention, but the following description is for easy understanding of the present invention, and the scope of the present invention is not limited to the following embodiments, and other embodiments obtained by appropriately replacing the configurations of the following embodiments are also included in the scope of the present invention.
[1] Description of the words
The term "fetal appendages" as used herein refers to the fetal membranes, placenta, umbilical cord and amniotic fluid. The term "fetal membrane" refers to a fetal sac containing amniotic fluid of a fetus and is formed from an amniotic membrane, a chorion, and an decidua membrane from the inside. Wherein the amniotic membrane and chorion originate from the fetus. By "amniotic membrane" is meant a blood vessel-deficient transparent membrane located on the innermost layer of the fetal membrane. The inner layer of the amniotic membrane (also referred to as epithelial cell layer) is coated with a layer of epithelial cells having a secretion function to secrete amniotic fluid, and the outer layer of the amniotic membrane (also referred to as extracellular matrix layer, corresponding to stroma) contains adherent stem cells.
The term "adherent stem cell" as used herein refers to a stem cell satisfying the following definition, "Mesenchymal stromal cells (mesenchyme stromal cells)" and "Mesenchymal stem cells (mesenchyme stem cells)" are also included in the adherent stem cell. In the present specification, "mesenchymal stem cell" is sometimes also referred to as "MSC".
Among adult stem cells (tissue stem cells) that can be collected from various tissues and organs, cells satisfying the following definition can be used as "adherent stem cells". Examples of the adult stem cells (tissue stem cells) include: bone marrow-derived mesenchymal stem cells, hematopoietic stem cells, stem cells in umbilical cord blood, umbilical cord-derived stem cells, amnion-derived stem cells, amniotic fluid stem cells, placental villus cell-derived mesenchymal stem cells, neural stem cells, adipose tissue-derived stem cells, pancreatic stem cells, synovial mesenchymal stem cells, dental pulp-derived stem cells derived from deciduous teeth, sperm stem cells (GS cells), testicular pluripotent stem cells (mGS cells), corneal epithelial stem cells, corneal parenchymal stem cells, pigment stem cells, tissue stem cells in organs, and the like, but are not particularly limited.
Definition of adherent Stem cells
i) Adherence to plastic was shown under culture conditions in standard medium.
ii) surface antigens CD73, CD90 are positive and CD326 is negative.
The "adherent stem cell" may satisfy the definition of i) ii) above, and the presence or absence of differentiation into bone, cartilage, fat, and the like is not particularly limited. The term "adherent stem cell" as used herein also includes cells having the ability to differentiate into bone, cartilage and fat, such as mesenchymal stem cells. The "adherent stem cells" also include cells that, although satisfying the above definition, do not have the ability to differentiate into bone, cartilage, or fat. In addition, the "adherent stem cells" also include cells that, although satisfying the above definition, differentiate into only any 1 or 2 of bone, cartilage, and fat.
The term "amnion-derived adherent stem cell" as used herein refers to an amnion-derived adherent stem cell, and includes "amnion mesenchymal stromal cell" and "amnion mesenchymal stem cell". In the present specification, "amniotic mesenchymal stem cells" are sometimes referred to as "amniotic MSCs".
The term "adherent stem cell group" as used herein refers to a cell group including adherent stem cells, and the form thereof is not particularly limited, and examples thereof include cell granules (cell pellet), cell sheets, cell aggregates, cell floating liquids, and cell suspensions.
The term "karyotypic abnormality" as used herein means structural abnormality of chromosome, including numerical abnormality and local structural abnormality of chromosome. As the number abnormality, there can be mentioned: there is usually only 1 "monosomy" of 2 paired chromosomes, or a "trisomy" of 3 chromosomes. As the local structural abnormality, there can be mentioned: translocation, inversion, deletion.
The term "normal karyotype" as used herein means a karyotype in which the above-mentioned karyotype abnormality or a karyotype close to normal is not observed.
"Normal karyotypes" and "karyotypic abnormalities" can be assessed by karyotyping. Specifically, each chromosome is identified based on a characteristic band pattern of the chromosome detected by a banding technique (differential staining technique), and quantitative abnormalities, local structural abnormalities, and the like are analyzed, whereby "normal karyotype" and "karyotype abnormalities" can be evaluated. The type of karyotype analysis is not particularly limited, and Q-band analysis using fluorescent dyes such as quinacrine mustard and Hoechst, G-band analysis using proteolytic enzyme treatment such as trypsin and giemsa staining (giemsa staining), analysis using multicolor-FISH method in which all chromosomes are stained in different colors, simple analysis using giemsa staining, and the like are known, and any method can be used in the present invention. For example, in order to determine a karyotypic abnormality, chromosomes may be extracted from 20 cells and subjected to karyotyping analysis to evaluate the presence or absence of a karyotypic abnormality. As a criterion for determining a normal karyotype, the proportion of cells having a karyotype abnormality among 20 cells to be analyzed is preferably 10% or less, more preferably 5% or less, further preferably 4% or less, further preferably 3% or less, further preferably 2% or less, further preferably 1% or less, further preferably 0%.
The term "ratio of adherent stem cells positive to KCNAB 1" as used herein refers to the ratio of cells positive to the antigen obtained by flow cytometry analysis as described in examples described below. In the present specification, the "ratio of cells positive for an antigen" is sometimes referred to as "positive rate".
[2] Cell populations comprising adherent stem cells
The cell population containing adherent stem cells is characterized in that the percentage of adherent stem cells positive for KCNAB1 in the cell population is 85% or more.
When the cell population containing adherent stem cells provided by the present invention satisfies the condition that the ratio of adherent stem cells positive for KCNAB1 is 85% or more, a cell population containing adherent stem cells that retain a normal karyotype is formed. Therefore, in the present invention, the above conditions can be used as an index for forming a cell population including adherent stem cells that retain a normal karyotype. Further, by measuring the above-mentioned index over time, it is possible to quickly and easily grasp and predict the change in karyotypic abnormality of the adherent stem cells. In addition, according to the present invention, by using the above-mentioned index, the quality of the donor itself and/or the biological sample collected from the donor can be evaluated. Further, according to the present invention, by using the above-mentioned index, it is possible to judge and/or predict whether or not the enzyme treatment method is optimal when the enzyme treatment is performed on the biological sample collected from the donor. In addition to the above-mentioned indices, the relative expression levels of the KCNAB1 gene, SULT1E1 gene, MN1 gene and RARRES2 gene in a specific range of values can be used as an index for the formation of a cell population including adherent stem cells that retain a normal karyotype.
In the cell population, the ratio of adherent stem cells positive to KCNAB1 is preferably 86% or more, more preferably 87% or more, further preferably 88% or more, further preferably 89% or more, further preferably 90% or more, further preferably 91% or more, further preferably 92% or more, further preferably 93% or more, further preferably 94% or more, further preferably 95% or more, further preferably 96% or more, further preferably 97% or more, further preferably 98% or more, further preferably 99% or more, and further preferably 100%.
According to one embodiment of the present invention, the population of cells comprising adherent stem cells provided by the present invention can satisfy a ratio of adherent stem cells positive for CD105, CD73, and/or CD90 of 90% or more.
CD105 refers to the differentiated population 105 and is a protein known as Endoglin.
CD73 refers to clade 73 and is a protein known as a 5-nucleotidase or Ecto-5' -nucleotidase.
CD90 refers to the clade 90 and is a protein known as Thy-1.
The percentage of adherent stem cells that are positive for CD105 in the cell population may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
The percentage of adherent stem cells positive for CD73 in the cell population may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
The percentage of adherent stem cells positive for CD90 in the cell population may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
According to one embodiment of the present invention, the population of cells including adherent stem cells provided by the present invention can satisfy a requirement that the rate of adherent stem cells positive for CD166 is 30% or more.
CD166 refers to a differentiated population 166, a protein known as activated leukocyte adhesion molecule (ALCAM).
The percentage of adherent stem cells that are positive for CD166 in the cell population may be 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
According to one embodiment of the present invention, the population of cells comprising adherent stem cells provided by the present invention can satisfy a ratio of adherent stem cells negative for CD45, CD34, and/or CD326 of 95% or more.
CD45 refers to the differentiation group 45 and is a protein known as PTPRC (protein tyrosine phosphatase, receptor type, C), or LCA (leukocyte common antigen).
CD34 refers to the differentiated population 34, a protein known as Hematographic promoter cell antigen CD 34.
CD326 refers to a differentiation group 326, a protein known as an epithelial cell adhesion molecule encoded by the EPCAM gene.
The percentage of adherent stem cells that are negative for CD45 in the cell population may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
The percentage of adherent stem cells that are negative for CD34 in the cell population may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
The percentage of adherent stem cells in the population that are negative for CD326 may be 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
Various antigens including KCNAB1, CD73, CD90, CD166, CD34, CD45, CD326 can be detected by any detection method known in the art. Examples of a method for detecting these antigens include: flow cytometry or cell staining, but is not limited thereto. In flow cytometry using a fluorescently labeled antibody, when a cell that fluoresces more strongly than a negative control (isotype control) is detected, the cell is judged to be "positive" for the marker. The fluorescently labeled antibody may be any antibody known in the art, and examples thereof include, but are not limited to, antibodies labeled with Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE), Allophycocyanin (APC), and the like. When a stained or fluorescent cell is observed under a microscope during cell staining, the cell is judged to be "positive" for the marker. The cell staining may be immune cell staining using an antibody or non-immune cell staining without using an antibody.
Specifically, the ratio of cells positive to KCNAB1 (positive rate) can be determined by the following steps (1) to (8) using dot-plot analysis by flow cytometry.
(1) The cryopreserved cell population was thawed and recovered by centrifugation. The recovered cell population was washed with Phosphate Buffered Saline (PBS) and recovered by centrifugation.
(2) After cell fixation/membrane permeabilization treatment using a solution of 4% paraformaldehyde with 0.1% polyoxyethylene (10) octylphenyl ether (Triton-X) at the final concentration, the cells were treatedCells were washed with Phosphate Buffered Saline (PBS) and 2.0 × 10 prepared with 0.5% BSA/PBS6Cell suspension per mL. The cell suspensions were dispensed in 100. mu.L each.
(3) The cell suspension after the distribution was centrifuged, and 100. mu.L of 0.5% BSA/PBS was added to each of the obtained cell pellets. Subsequently, an antibody corresponding to each antigen marker or an antibody for isotype control thereof is added. The reaction solutions were mixed by vortexing, and then allowed to stand at 4 ℃ for 20 minutes.
(4) 0.5% BSA/PBS was added, the cells were washed by centrifugation, and then suspended in 0.5% BSA/PBS, followed by filtration through a cell filter (35 μm nylon mesh filter) (Corning Co., Ltd./model: 352235).
(5) Using BD AccuriTMThe cell suspension (ALL Event 10000) obtained by filtration through a filter was analyzed by a C6 Flow Cytometer (Becton, Dickinson and Company).
(6) The measurement results were plotted as a dot diagram with SSC (side scattered light) on the vertical axis and FSC (front scattered light) on the horizontal axis.
(7) In the dot diagram, the entire region (gate) in which the fluorescence intensity of all cells measured with the antibody for isotype control is higher than 0.5% was selected.
(8) The ratio of cells contained in the gate selected in (7) among all the cells measured with the antibody corresponding to the antigen marker was calculated.
The ratio of cells negative to each surface antigen (negative rate) was calculated by the following formula.
The percent of negative results (%) -100-positive results
The timing for detecting the KCNAB1 is not particularly limited, and examples thereof include: the present invention is not limited to the above-described examples, and the examples include the following examples, such as immediately after the isolation of cells from a biological sample, during the culture step, after purification in the culture step, immediately after n passages (n represents an integer of 1 or more), during the maintenance of culture, before cryopreservation, after thawing, or before the preparation becomes a pharmaceutical composition.
The cell population containing adherent stem cells provided by the present invention preferably satisfies a relative expression level of the KCNAB1 gene with respect to the expression level of the SDHA gene of 0.05 or more.
The relative expression amount of the KCNAB1 gene with respect to the expression amount of the SDHA gene may be 0.75 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.25 or more, 0.3 or more, 0.35 or more, or 0.4 or more. The upper limit of the relative expression amount of the KCNAB1 gene with respect to the expression amount of the SDHA gene is not particularly limited, and may be, for example, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, 0.9 or less, 0.8 or less, 0.7 or less, or 0.6 or less.
The cell population containing adherent stem cells provided by the present invention preferably satisfies a relative expression level of SULT1E1 gene with respect to the expression level of SDHA gene of 0.1 or more.
The relative expression amount of SULT1E1 gene with respect to the expression amount of the SDHA gene may be 0.13 or more, 0.15 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, or 0.6 or more. The upper limit of the relative expression amount of SULT1E1 gene with respect to the expression amount of the SDHA gene is not particularly limited, and may be, for example, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, or 0.5 or less.
The cell population containing adherent stem cells provided by the present invention preferably satisfies that the relative expression level of MN1 gene with respect to the expression level of SDHA gene is 0.7 or more.
The relative expression level of MN1 gene with respect to the expression level of SDHA gene may be 0.8 or more, 0.9 or more, 1 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, or 1.7 or more. The upper limit of the relative expression amount of MN1 gene with respect to the expression amount of SDHA gene is not particularly limited, and may be, for example, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
The cell population containing adherent stem cells provided by the present invention preferably satisfies that the relative expression level of the RARRES2 gene with respect to the expression level of the SDHA gene is 0.4 or less.
The relative expression level of the RARRES2 gene with respect to the expression level of the SDHA gene may be 0.3 or less, 0.2 or less, 0.1 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.009 or less, 0.008 or less, 0.007 or less, 0.006 or less, 0.005 or less, 0.004 or less, or 0.003 or less. The lower limit of the relative expression level of the RARRES2 gene with respect to the expression level of the SDHA gene is not particularly limited, and may be, for example, 0.001 or more, or 0.002 or more.
The method for detecting each gene and/or measuring the expression level thereof is not particularly limited as long as it is a method known in the art, and, for example, microarray (microarray), RT-PCR, quantitative RT-PCR, or Northern blot hybridization can be used. As a method for measuring the relative expression amount of each gene with respect to the expression amount of the SDHA gene, a microarray can be used. Specifically, the microarray can be performed according to the following steps (1) to (5). The following steps (3) to (5) may be carried out by RIKEN GENESIS.
(1) The cryopreserved cell population was thawed and recovered by centrifugation. The recovered cell population was washed with Phosphate Buffered Saline (PBS), and the cells were recovered by centrifugation.
(2) Total RNA was extracted and purified using an RNA extraction kit (RNeasy Plus Mini kit (QIAGEN)).
(3) Using the purified total RNA as a template, cDNA was synthesized by reverse transcription, and further transcribed from the synthesized cDNA to cRNA by in vitro transcription, and biotin labeling was performed.
(4) Biotin-labeled cRNA was added to the hybridization buffer, and hybridization was performed for 16 hours on a Human Genegenome U133A2.0array (manufactured by Affymetrix). Washing was performed with a GeneChip fluids Station 450 (manufactured by Affymetrix), after phycoerythrin staining, scanning was performed with a GeneChip Scanner 30007G (manufactured by Affymetrix), image analysis was performed with agcc (Affymetrix GeneChip Command conditioner software) (manufactured by Affymetrix), and digitization was performed with an Affymetrix Expression conditioner (manufactured by Affymetrix).
(5) The numerical data files were compared and analyzed using analysis software GeneSpring GX (Agilent Technologies, Inc.). The relative expression level of each gene in each cell with respect to the expression level of the SDHA gene was calculated.
Quantitative PCR can be used as a method for measuring the relative expression level of each gene with respect to the expression level of the SDHA gene. The quantitative PCR can be specifically carried out according to the following steps (1) to (5).
(1) The cryopreserved cell population was thawed and recovered by centrifugation. The recovered cell population was washed with Phosphate Buffered Saline (PBS), and the cells were recovered by centrifugation.
(2) Total RNA was extracted and purified using an RNA extraction kit (RNeasy Plus Mini kit (QIAGEN)).
(3) cDNA was synthesized by reverse transcription using purified total RNA as a template, and the synthesized cDNA, Taqman Fast Advanced Master Mix (Applied Biosystems) and primers (Taqman Gene Expression Assay, Thermo Fisher) were mixed and injected into a 96-well plate to perform quantitative PCR.
(4) The delta Ct value of each sample for SDHA was analyzed by the StepOneNus Real-Time PCR System (Applied Biosystems Co., Ltd.), and the relative expression level (2^ (-delta Ct)) of each gene in each cell relative to the expression level of SDHA gene was calculated.
SDHA (succinate dehydrogenase complex, subunit A) is one of the housekeeping genes, the sequence of which is registered in the gene database of the National Center for Biotechnology Information (National Center for Biotechnology Information) as ID: 6389. SDHA is a gene comprising the base sequence represented by SEQ ID NO. 1 or a gene encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO. 2.
The sequence of KCNAB1 (potassium channel, voltage gated subset a regulatory beta subbentt 1) gene was registered as ID in the gene database of the national center for biotechnology information: 7881. KCNAB1 is a gene consisting of the base sequence shown in SEQ ID NO. 3 or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 4.
The sequence of the SULT1E1 (sulfotransferase family 1E member 1) gene was registered in the gene database of the national center for biotechnology information as ID: 6783. SULT1E1 is a gene comprising the base sequence represented by SEQ ID NO. 5 or a gene encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO. 6.
The sequence of MN1 (meningioma (balanced translocation disrupted) 1) gene was registered as ID: 4330. MN1 is a gene comprising the base sequence represented by SEQ ID NO. 7 or a gene encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO. 8.
The sequence of RARRES2 (retinoic acid receptor responsive protein (tazarotene induced)2) gene was registered as ID: 5919. RARES 2 is a gene comprising the base sequence represented by SEQ ID NO. 9 or a gene encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO. 10.
The timing for measuring the gene expression level is not particularly limited, and examples thereof include: the present invention is not limited to the above-described examples, and the examples include the following examples, such as immediately after the isolation of cells from a biological sample, during the culture step, after purification in the culture step, immediately after n passages (n represents an integer of 1 or more), during the maintenance of culture, before cryopreservation, after thawing, or before the preparation becomes a pharmaceutical composition.
In the cell population of the present invention, the adherent stem cells may be cultured to maintain a normal karyotype without stopping proliferation to preferably 20 days later, more preferably 25 days later, 30 days later, 35 days later, 40 days later, 45 days later, 50 days later, 55 days later, 60 days later, 65 days later, 70 days later, 75 days later, 80 days later, 85 days later, 90 days later, 95 days later, 100 days later, 105 days later, or 110 days later after the start of in vitro culture.
The adherent stem cells in the cell population of the present invention may be cultured until the passable number is 1 or more, preferably 2 or more, more preferably 3 or more, further preferably 4 or more, further preferably 5 or more, further preferably 6 or more, further preferably 8 or more, further preferably 10 or more, further preferably 12 or more, further preferably 14 or more, further preferably 16 or more, further preferably 18 or more, further preferably 20 or more, further preferably 22 or more, further preferably 24 or more, and further preferably 25 or more while maintaining the normal karyotype. The upper limit of the passable number is not particularly limited, and is, for example, 50 times or less, 45 times or less, 40 times or less, 35 times or less, or 30 times or less.
The cell population comprising adherent stem cells provided by the present invention can be multiplied preferably 10 times or more, more preferably 20 times or more, 30 times or more, 40 times or more, 50 times or more, or 60 times or more while maintaining a normal karyotype. The cell population including adherent stem cells provided by the present invention may be multiplied by, for example, 100 times or less, 90 times or less, 80 times or less, or 70 times or less, but is not limited thereto. The number of population doublings is the number of cell population divisions in a given culture period and can be determined by log10(number of cells at the end of culture) -log10(number of cells at the beginning of culture)]/log10(2) The calculation formula (2) is calculated. When subculture is performed, the total population doubling time is calculated by calculating the population doubling time for each passage using the above formula and then accumulating the population doubling times.
The cell survival rate of the cell population comprising adherent stem cells provided by the present invention can be determined by trypan blue staining, PI (propidium iodide) staining, MTT (3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium, for example
Figure BDA0002557821570000151
Bromide) test, etc., but is not limited thereto.
The cell survival rate of the cell population including adherent stem cells provided by the present invention is preferably 70% or more, and more preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
The source of the adherent stem cells is not particularly limited, and, for example, adherent stem cells derived from a fetal appendage, bone marrow, adipose tissue, or dental pulp can be used. The adherent stem cells are preferably adherent stem cells from a fetal appendage, more preferably adherent stem cells from an amniotic membrane. Adherent stem cells are adherent stem cells isolated from a sample of an autologous, allogeneic or xenogeneic organism, preferably adherent stem cells isolated from a sample of an allogeneic organism.
The adherent stem cells are adherent stem cells subjected to or not subjected to gene recombination, and preferably are adherent stem cells not subjected to gene recombination.
The cell population of the invention can comprise any number of adherent stem cells the cell population of the invention can comprise, for example, 1.0 × 1011.0 × 1021.0 × 1031.0 × 1041.0 × 1051.0 × 1061.0 × 1071.0 × 1081.0 × 1091.0 × 10101.0 × 10111.0 × 10121.0 × 1013More or less adherent stem cells, but not limited thereto.
The cell population of the present invention may contain an arbitrary component in addition to the adherent stem cells. Examples of the above-mentioned components include: salts (e.g., physiological saline, ringer's solution, BICANATE infusion), polysaccharides (e.g., hydroxyethyl starch (HES), dextran, etc.), proteins (e.g., albumin, etc.), dimethyl sulfoxide (DMSO), amino acids, medium components (e.g., components contained in RPMI1640 medium), etc., but are not limited thereto.
The cell population of the present invention may be stored in a frozen state until just before use. The cell population may contain a cryopreservation solution in addition to the adherent stem cells. As the cryopreservation solution, a commercially available cryopreservation solution can be used. Examples thereof include: CP-1 (registered trademark) (manufactured by Toyobo pharmaceutical industries Co., Ltd.), BAMBANKER (manufactured by Lymphotec Co., Ltd.), STEM-CELLBANKER (manufactured by Nippon Kagaku K.K.), Reprocryo RM (manufactured by REPROELL Co., Ltd.), CryoNovo (manufactured by Akron Biotechnology Co., Ltd.), MSC Freezing Solution (manufactured by biologicals industries Co., Ltd.), CryoStor (manufactured by HemaCare Co., Ltd.), and the like, but are not limited thereto.
The cell population of the present invention may be provided in the form of a composition combined with a medium. As the medium, a liquid medium (for example, a culture medium, dimethyl sulfoxide (DMSO), a cryopreservation solution, or a pharmaceutically acceptable vehicle described later) can be preferably used.
The cell concentration of the composition comprising the cell population of the invention and the mediator may be, for example, 1.0 × 1011.0 × 10/mL21.0 × 10/mL31.0 × 10/mL41.0 × 10/mL51.0 × 10/mL61.0 × 10/mL71.0 × 10/mL81.0 × 10/mL91.0 × 10/mL10The cell concentration is not less than or equal to one/mL, but is not limited thereto.
[3] Method for producing cell population comprising adherent stem cells
The method for producing a cell population comprising adherent stem cells of the present invention comprises: a cell population containing cells collected from a biological tissue or organ such as a fetal appendage is obtained, and the ratio of adherent stem cells positive for KCNAB1 in the cell population is 85% or more. The method for producing a cell population containing adherent stem cells of the present invention comprises: culturing a cell population containing cells collected from a biological tissue or organ such as a fetal appendage under conditions that maintain a ratio of adherent stem cells positive for KCNAB1 of 85% or more. The above conditions are an index for the formation of a cell population including adherent stem cells that retain a normal karyotype, and the culture method of the present invention is not particularly limited as long as the above index is satisfied.
The manufacturing method of the present invention may include, for example: a cell population obtaining step of obtaining a cell population including adherent stem cells by subjecting a fetal appendage such as amniotic membrane to an enzyme treatment. The above-mentioned cell population obtaining step may include a step of obtaining an amnion by a caesarean section. The cell population obtaining step may include a step of washing a biological sample containing adherent stem cells.
The amniotic membrane is composed of an epithelial cell layer and an extracellular matrix layer, the latter comprising amnion adherent stem cells. The amniotic epithelial cells are characterized by expressing epithelial adhesion factor (EpCAM: CD326) as in other epithelial cells, whereas the amniotic adherent stem cells do not express the epithelial-specific surface antigen marker for CD326 and can be readily differentiated by flow cytometry. The above-mentioned cell population obtaining step may include a step of obtaining an amnion by a caesarean section.
The cell population comprising adherent stem cells of the present invention is preferably a cell population obtained by treating a biological sample comprising an epithelial cell layer and an adherent stem cell layer collected from a fetal appendage with at least collagenase.
The enzymatic treatment of a biological sample collected from a fetal appendage, preferably a biological sample comprising an epithelial cell layer and an adherent stem cell layer, is preferably a treatment with an enzyme (or a combination thereof) that is capable of freeing adherent stem cells comprised in the extracellular matrix layer of the fetal appendage and that does not break down the epithelial cell layer. Such an enzyme is not particularly limited, and examples thereof include collagenase and/or metalloprotease. Examples of the metalloprotease include thermolysin and/or Dispase (Dispase) which are metalloproteases cleaving the N-terminal side of the nonpolar amino acid, and are not particularly limited.
The collagenase activity concentration is preferably 50PU/ml or more, more preferably 100PU/ml or more, and still more preferably 200PU/ml or more. The collagenase activity concentration is not particularly limited, but is, for example, 1000PU/ml or less, 900PU/ml or less, 800PU/ml or less, 700PU/ml or less, 600PU/ml or less, or 500PU/ml or less. Here, PU (protease Unit) is defined as the amount of enzyme that decomposes 1ug of FITC-collagen at pH7.5 at 30 ℃ for 1 minute.
The activity concentration of the metalloprotease (e.g., thermolysin and/or Dispase) is preferably 50PU/ml or more, more preferably 100PU/ml or more, and still more preferably 200PU/ml or more. The active concentration of the metalloprotease is preferably 1000PU/ml or less, more preferably 900PU/ml or less, further preferably 800PU/ml or less, further preferably 700PU/ml or less, further preferably 600PU/ml or less, further preferably 500PU/ml or less. Here, in the embodiment using Dispase (Dispase) as the metalloprotease, PU (protease Unit) is defined as the amount of enzyme that releases 1ug of tyrosine-equivalent amino acid from casein (casein lactate) at pH7.5 at 30 ℃ for 1 minute. Within the above-mentioned range of enzyme concentration, it is possible to prevent the contamination of epithelial cells contained in the epithelial cell layer of the fetal appendage and to efficiently release adherent stem cells contained in the extracellular matrix layer. The preferred combination of concentrations of collagenase and/or metalloprotease can be determined by microscopic observation of the enzyme-treated fetal appendages, flow cytometry of the cells obtained.
From the viewpoint of efficiently recovering living cells, it is preferable to treat the fetal appendages with a combination of collagenase and metalloprotease. It is further preferred that the fetal appendages are treated simultaneously by the above combination. In this case, thermolysin and/or Dispase (Dispase) can be used as the metalloprotease, but the metalloprotease is not limited thereto. Adherent stem cells can be obtained simply by treating a fetal appendage with an enzyme solution containing collagenase and metalloprotease only once. In addition, the risk of contamination with bacteria, viruses, and the like can be reduced by performing the treatment at the same time.
The enzymatic treatment of fetal appendages is preferably carried out by immersing amnion washed with a washing solution such as physiological saline or Hank's balanced salt solution in an enzymatic solution and stirring the amnion with a stirring device. As such a stirring device, for example, a stirrer or a vibrator can be used from the viewpoint of efficiently releasing the adherent stem cells contained in the extracellular matrix layer of the fetal appendage, but the stirring device is not limited thereto. The stirring speed is not particularly limited, and when a stirrer or a vibrator is used, it is, for example, 10rpm or more, 30rpm or more, or 50rpm or more. The stirring speed is not particularly limited, and when a stirrer or a vibrator is used, it is, for example, 100rpm or less, 80rpm or less, or 60rpm or less. The enzyme treatment time is not particularly limited, and is, for example, 10 minutes or more, 30 minutes or more, 50 minutes or more, 70 minutes or more, or 90 minutes or more. The enzyme treatment time is not particularly limited, and is, for example, 6 hours or less, 4 hours or less, 2 hours or less, and 100 minutes or less. The enzyme treatment temperature is not particularly limited, and is, for example, 16 ℃ or higher, 20 ℃ or higher, 24 ℃ or higher, 28 ℃ or higher, 32 ℃ or higher, or 36 ℃ or higher. The enzyme treatment temperature is not particularly limited, and is, for example, 40 ℃ or lower, 39 ℃ or lower, or 38 ℃ or lower.
In the production method of the present invention, the free adherent stem cells can be separated and/or recovered from the enzyme solution containing the free adherent stem cells by a known method such as a filter, centrifugation, a hollow fiber separation membrane, or a cell sorter, as desired. The enzyme solution comprising free adherent stem cells is preferably filtered using a filter. In the method of filtering the enzyme solution with a filter, only the free cells are passed through the filter, and the undecomposed epithelial cell layer remains on the filter without passing through the filter, so that not only can the free adherent stem cells be easily separated and/or recovered, but also the risk of contamination with bacteria, viruses, and the like can be reduced. The filter is not particularly limited, and examples thereof include a mesh filter. The pore diameter (size of mesh) of the mesh filter is not particularly limited, and is, for example, 40 μm or more, 60 μm or more, 80 μm or more, or 90 μm or more. The pore size of the mesh filter is not particularly limited, but is, for example, 200 μm or less, 180 μm or less, 160 μm or less, 140 μm or less, 120 μm or less, or 100 μm or less. The filtration rate is not particularly limited, and when the pore diameter of the mesh filter is set to the above range, the enzyme solution containing adherent stem cells can be filtered by allowing it to naturally fall, thereby preventing the decrease in cell survival rate.
As a material of the mesh filter, nylon is preferably used. Tubes having a nylon mesh filter of 40 μm, 70 μm, 95 μm or 100 μm, such as Falcon cell filters, which are widely used for research purposes, can be used. Medical mesh cloths (nylon and polyester) used for hemodialysis and the like can be used. Further, an arterial filter (polyester mesh filter, pore size: 40 μm to 120 μm) used in extracorporeal circulation may be used. Other materials, such as stainless steel mesh filters, etc., may also be used.
When the adherent stem cells are passed through the filter, they are preferably allowed to drip (fall freely). A forced passage of a filter such as suction using a pump or the like may be used, and a pressure as low as possible is preferable in order to avoid damage to cells.
Adherent stem cells passed through the filter can be recovered by centrifugation after diluting the filtrate with a double or more amount of the medium or the balanced salt buffer. Examples of the balanced salt buffer include, but are not limited to, physiological saline, Dulbecco's Phosphate Buffer (DPBS), Earle's Balanced Salt Solution (EBSS), Hank's Balanced Salt Solution (HBSS), and Phosphate Buffer Solution (PBS).
The cell population obtained in the above-mentioned cell population obtaining step is cultured under the condition that the ratio of adherent stem cells positive for KCNAB1 in the above-mentioned cell population is 85% or more. The above conditions are useful as an index for obtaining a cell population containing adherent stem cells that retain a normal karyotype. The culture method is not particularly limited as long as the above-mentioned index is satisfied. Examples of such a method include a method in which a cell population satisfying the above-described index is obtained by a cell sorter and the cell population is cultured under the condition satisfying the above-described index.
The culture method satisfying the above-mentioned criteria includes, for example, repeating the culture at a cell density of 100 to 20,000 cells/cm2For example, the step of inoculating the cell population in an uncoated plastic culture vessel and culturing the cell population. The lower limit of the density at the time of seeding of the cell population is more preferably 200 cells/cm2Above, more preferably 400 pieces/cm2More preferably 600 pieces/cm2Above, more preferably 800 pieces/cm2More preferably 1000 pieces/cm2More preferably 1200 pieces/cm2The number of the molecules is more preferably 1400/cm2More preferably 1600/cm2Above, more preferably 1800 pieces/cm2More preferably 2000 pieces/cm2The above. The upper limit of the density at the time of seeding the cell population is more preferableIs 18000 pieces/cm2More preferably 16000 pieces/cm2More preferably 14000 cells/cm2Hereinafter, more preferably 12000 pieces/cm2Hereinafter, 10000 pieces/cm are more preferable2Hereinafter, more preferably 8000 pieces/cm2The following.
As another culturing method satisfying the above-mentioned criteria, for example, a method of repeating the culturing at 100 to 20,000 cells/cm2The step of culturing the cells by inoculating the cell population in a culture vessel made of plastic coated with a coating agent. The preferred conditions for the density at the time of seeding the cell population are the same as those described above.
Examples of the coating agent include: extracellular matrix, fibronectin, vitronectin, osteopontin, laminin, entactin (entactin), collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, gelatin, poly-L-ornithine, poly-D-lysine, Matrigel (registered trademark) matrix, but is not limited thereto.
The medium used for the above culture can be prepared by using an arbitrary liquid medium for animal cell culture as a basal medium and adding other components (albumin, serum-replacement reagent, growth factor, human platelet lysate, etc.) as needed.
As the basal Medium, there may be used, but not particularly limited to, BME Medium, BGJb Medium, CMRL1066 Medium, GlasgowMEM Medium, Improved MEM stretch Medium, IMDM Medium (Iscove's Modified Dulbecco's Medium), Medium 199 Medium, Eagle MEM Medium, alpha MEM (alpha MEM) Medium, DMEM Medium (Dulbecco's Modified Eagle's Medium), Ham's F10 Medium, Ham's F12 Medium, RPMI1640 Medium, Fischer's Medium, and mixed Medium thereof (for example, DMEM/F12 Medium (Dulbecco's Modified Eagle's Nuxture F-12 m)).
The medium used for the above culture may be a commercially available serum-free medium. Examples thereof include: STK1, STK2(DS PHARMA BIOMEDICAL Co., Ltd.), EXPREP MSC Medium (BioMimetics Symphies Co., Ltd.), Corning stemgro human adherent stem cell culture Medium (Corning Co., Ltd.), and the like, but are not particularly limited.
Examples of other components to be added to the basic medium include: albumin, serum replacement agents, growth factors, or human platelet lysate, and the like. In the above-described mode of adding albumin to the basal medium, the concentration of albumin is preferably more than 0.05% and 5% or less. In the above embodiment in which serum is added to the basal medium, the serum concentration is preferably 5% or more. In the case of the mode of adding the growth factor, a reagent (protein such as heparin, gel, polysaccharide, or the like) for stabilizing the growth factor in the culture medium may be further added in addition to the growth factor, or the stabilized growth factor may be previously added to the basic culture medium. Examples of the growth factor include, but are not limited to, Fibroblast Growth Factor (FGF), epithelial cell growth factor (EGF), Transforming Growth Factor (TGF), Vascular Endothelial Growth Factor (VEGF), platelet-derived growth factor (PDGF), and families thereof.
As another culture method satisfying the above-mentioned criteria, for example, a culture is carried out by adding human platelet lysate (hPL) to a basal medium used for the culture. The human platelet lysate is preferably subjected to inactivation of bacteria, viruses and/or sterilization treatment. As the human platelet lysate, a commercially available human platelet lysate can be used. Examples thereof include: stemulate (Cook Regentec), PLTMax (Mill Creek Life Science), UltraGRO (Aventacell BioMedial), PLUS (Compass Biomedical), etc., but the present invention is not limited thereto.
The final concentration of the human platelet lysate in the culture medium is preferably 1% or more, more preferably 2% or more, further preferably 3% or more, further preferably 4% or more, and further preferably 5% or more. The final concentration of the platelet lysate in the culture medium is preferably 20% or less, more preferably 18% or less, more preferably 16% or less, more preferably 14% or less, more preferably 12% or less, more preferably 10% or less, more preferably 9% or less, more preferably 8% or less, more preferably 7% or less, and more preferably 6% or less.
The timing of adding the human platelet lysate is not particularly limited, and examples thereof include: the culture medium may be used in the early stage of the culture step, during the culture step, after purification in the culture step, immediately after n passages (where n represents an integer of 1 or more), during the maintenance culture, before cryopreservation, after thawing, or the like.
The culture of adherent stem cells can be performed, for example, in the following steps. First, the cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a medium. Then, the cells were inoculated into a plastic culture vessel and the amount of CO was 3% to 5%2The concentration, 37 ℃ environment, using culture medium culture, so that the confluency is 95% or less, as the medium, for example, α MEM, M199, or based on them medium, but not limited to, this, through the culture of the cells obtained is 1 after the culture.
The culture time of the above-mentioned 1 culture may be, for example, 2 to 15 days, and more specifically, 2 days, 3 days, 4 days, 5 days, 6 days, 8 days, 10 days, 12 days, 14 days, or 15 days.
The cells after the 1-time culture can be further passaged and cultured as described below, for example. First, the cells after 1 culture were treated by a cell detaching method and detached from the plastic culture container. Subsequently, the resulting cell suspension was centrifuged to remove the supernatant, and the resulting cell pellet was suspended in a medium. Finally, the cells were inoculated into a plastic culture vessel containing 3% to 5% CO2The cells are cultured at a concentration of 37 ℃ in a medium such that the confluency is 95% or less, and examples of the medium include, but are not limited to, α MEM, M199, and a medium based thereon, and the cells obtained by the above-mentioned passaging and culturing are cultured after 1 passageThe cell of (1). By carrying out the same passaging and culturing, cells after n passages (n represents an integer of 1 or more) can be obtained. From the viewpoint of producing a large number of cells, the lower limit of the number of passages n is, for example, 1 or more, preferably 2 or more, more preferably 4 or more, further preferably 6 or more, further preferably 8 or more, further preferably 10 or more, further preferably 12 or more, further preferably 14 or more, further preferably 16 or more, further preferably 18 or more, further preferably 20 or more, and further preferably 25 or more. From the viewpoint of suppressing cell aging, the upper limit of the number of passages n is preferably 50 or less, 40 or less, or 30 or less, for example. As the cell separation method, for example, a cell separation agent can be used. Examples of the cell-releasing agent include trypsin, collagenase, Dispase (Dispase), ethylenediaminetetraacetic acid (EDTA), and the like, and are not particularly limited. As the cell-releasing agent, a commercially available cell-releasing agent can be used. Examples thereof include: examples of the trypsin-EDTA solution include, but are not limited to, trypsin-EDTA solution (manufactured by Thermo Fisher Scientific Co., Ltd.), TrypLE Select (manufactured by Thermo Fisher Scientific Co., Ltd.), Accutase (manufactured by Stemcell Technologies Co., Ltd.), and Accumax (manufactured by Stemcell Technologies Co., Ltd.). As the cell separation method, a physical cell separation method may be used, and for example, a cell scraper (manufactured by Corning) may be used. The cell separation method may be used alone or in combination of two or more.
According to the production method of the present invention, adherent stem cells retaining normal karyotype can be obtained, and thus a safe cell preparation (pharmaceutical composition) can be produced, the lower limit of the number of cells obtained per 1 culture batch (the number of cells obtained per unit surface area and per unit number of culture days) differs depending on the number of seeded cells, seeding density, and the like, and is, for example, 5.0 × 103(pieces/cm)2Day) above 6.0 × 103(pieces/cm)2/day) above 8.0 × 103(pieces/cm)2/day) above 1.0 × 104(pieces/cm)2/day) above 1.1 × 104(pieces/cm)2/day) or more, or 1.2 × 104(pieces/cm)2Day) or more, and the upper limit of the number of cells obtained per 1 culture batch is not particularly limited, and is, for example, 1.0 × 105(pieces/cm)2Day) below 8.0 × 104(pieces/cm)2Day) of less than 6.0 × 104(pieces/cm)2Day) of less than 4.0 × 104(pieces/cm)2/day) or less, or 2.0 × 104(pieces/cm)2And/day) below.
According to the production method of the present invention, adherent stem cells having a normal karyotype retained can be obtained. Thus, the adherent stem cells obtained by the production method of the present invention can be cultured, after the start of in vitro culture, to maintain a normal karyotype without stopping proliferation, preferably for 20 days, more preferably for 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, or 110 days.
Furthermore, the adherent stem cells obtained by the production method of the present invention can be cultured to the number of doublings, preferably 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 60 or more, while maintaining the normal karyotype and without stopping the proliferation after the start of in vitro culture.
The manufacturing method of the present invention may include a recognition process including: identifying a population containing adherent stem cells that retain a normal karyotype, using as an indicator that the percentage of adherent stem cells that are positive for KCNAB1 in the population containing adherent stem cells is 85% or more.
The method for identifying a cell population comprising the aforementioned adherent stem cells is preferably flow cytometry, microarray, RT-PCR, and/or quantitative RT-PCR.
The timing of the above recognition is not particularly limited, and examples thereof include: the present invention is not limited to the above-described examples, and the examples include the following examples, such as immediately after the isolation of cells from a biological sample, during the culture step, after purification in the culture step, immediately after n passages (n represents an integer of 1 or more), during the maintenance of culture, before cryopreservation, after thawing, or before the preparation becomes a pharmaceutical composition.
The manufacturing method of the present invention may further include: after identifying a cell population containing the aforementioned adherent stem cells using as an indicator that the percentage of adherent stem cells positive for KCNAB1 is 85% or more, the identified cell population is selectively isolated. The method for selectively separating the identified cell population is not particularly limited, and examples thereof include: sorting of cell groups by a cell sorter, purification of cell groups by culture, and the like.
In addition, the manufacturing method of the present invention may include: and (d) performing cryopreservation of the cell population containing the adherent stem cells. In the embodiment including the step of cryopreserving the cell population, the cell population may be separated, collected, and/or cultured as necessary after thawing the cell population. Alternatively, the cell population may be used as it is after thawing.
The method for cryopreservation of a cell population containing the aforementioned adherent stem cells is not particularly limited, and examples thereof include: program freezers, deep freezers, immersion in liquid nitrogen, and the like. The temperature during freezing is preferably-30 deg.C or lower, -40 deg.C or lower, -50 deg.C or lower, -80 deg.C or lower, -90 deg.C or lower, -100 deg.C or lower, -150 deg.C or lower, -180 deg.C or lower, or-196 deg.C or lower (liquid nitrogen temperature). The preferred freezing rate for freezing is, for example, -1 ℃/min, -2 ℃/min, -5 ℃/min, -9 ℃/min, -10 ℃/min, -11 ℃/min, or-15 ℃/min. In the case of using a program freezer as the freezing method, the temperature can be stably lowered to a temperature between-50 ℃ and-30 ℃ (for example, -40 ℃) at a freezing rate of-2 ℃/min or more and-1 ℃/min or less, and further lowered to a temperature between-100 ℃ and-80 ℃ (for example, -90 ℃) at a freezing rate of-11 ℃/min or more and-9 ℃/min or less (for example, -10 ℃/min). In the case of using the above freezing method by immersing in liquid nitrogen, for example, the temperature may be rapidly lowered to-196 ℃ to freeze the sample, and then the sample may be cryopreserved in liquid nitrogen (gas phase).
When the freezing method is used for freezing, the cell population may be frozen in a state of being placed in an arbitrary storage container. Examples of such a storage container include: a vial for cryopreservation, a bag for freezing, an infusion bag, etc., but the present invention is not limited thereto.
When the freezing method is employed, the cell population may be frozen in an arbitrary cryopreservation solution. As the cryopreservation solution, a commercially available cryopreservation solution can be used. Examples thereof include: CP-1 (registered trademark) (manufactured by Toyobo pharmaceutical Industries Co., Ltd.), BAMBANKER (manufactured by Lymphotec Co., Ltd.), STEM-CELLBANKER (manufactured by Nippon Kagaku K.K.), Reprocryo RM (manufactured by REPROELL Co., Ltd.), CryoNovo (manufactured by Akron Biotechnology Co., Ltd.), MSC Freezing Solution (manufactured by Biological Industries Co., Ltd.), CryoStor (manufactured by HemaCare Co., Ltd.), and the like, but are not limited thereto.
The cryopreservation solution may contain a polysaccharide at a predetermined concentration. The preferable concentration of the polysaccharide is, for example, 1 mass% or more, 2 mass% or more, 4 mass% or more, or 6 mass% or more. The preferable concentration of the polysaccharide is, for example, 20 mass% or less, 18 mass% or less, 16 mass% or less, 14 mass% or less, or 13 mass% or less. Examples of the polysaccharide include, but are not limited to, hydroxyethyl starch (HES) and Dextran (Dextran 40).
The cryopreservation solution may contain dimethyl sulfoxide (DMSO) at a given concentration. The preferable concentration of DMSO is, for example, 1 mass% or more, 2 mass% or more, 3 mass% or more, 4 mass% or more, or 5 mass% or more. Further, the preferable concentration of DMSO is, for example, 20 mass% or less, 18 mass% or less, 16 mass% or less, 14 mass% or less, 12 mass% or less, or 10 mass% or less.
The cryopreservation solution may contain albumin at a given concentration of more than 0 mass%. The preferable concentration of albumin is, for example, 1 mass% or more, 2 mass% or more, 3 mass% or more, or 4 mass% or more. The preferable concentration of albumin is, for example, 30 mass% or less, 20 mass% or less, 10 mass% or less, or 9 mass% or less. Examples of albumin include: bovine Serum Albumin (BSA), mouse albumin, human albumin, and the like, but is not limited thereto.
The manufacturing method of the present invention may include: and washing the cell population containing the adherent stem cells. Examples of the cleaning solution used in the step of cleaning the cell population containing the adherent stem cells include: physiological saline, Dulbecco's Phosphate Buffered Saline (DPBS), Earle's Balanced Salt Solution (EBSS), Hank's Balanced Salt Solution (HBSS), Phosphate Buffered Saline (PBS), and the like, but is not limited thereto. By washing the cell population, allergens, endotoxins, and the like can be reduced or removed. Examples of the above-mentioned allergens include: bovine Serum Albumin (BSA), porcine trypsin, porcine heparin, and the like, but is not limited thereto.
The manufacturing method of the present invention may include: and (d) removing, if necessary, undesired cell aggregates from the cell population containing the adherent stem cells. The step of removing undesired cell aggregates from a cell population comprising the aforementioned adherent stem cells may comprise: a step of filtering a cell population (cell suspension) containing adherent stem cells with a filter.
The manufacturing method of the present invention may include: and filling a storage container with a cell population containing the adherent stem cells. Examples of the storage container include: a vial for cryopreservation, a bag for freezing, an infusion bag, etc., but the present invention is not limited thereto.
[4] Method for monitoring karyotypic abnormality of adherent stem cells, method for evaluating donor and/or biological sample collected from donor, and method for judging and/or predicting optimal enzyme treatment conditions
In the present invention, karyotypic abnormalities of adherent stem cells can be monitored by measuring (preferably by measurement over time) the percentage of adherent stem cells positive for KCNAB1 in a cell population containing adherent stem cells as an indicator of 85% or more. Examples of the steps necessary for the monitoring include a step of performing culture, a step of performing cryopreservation, and/or a step of performing a preparation.
By measuring the index over time in the step of culturing, the change in karyotypic abnormality of the adherent stem cells can be quickly and easily grasped and predicted. It is known that adherent stem cells maintain normal karyotype in a cell population including adherent stem cells that satisfy the above criteria. On the other hand, when the culture state with a value exceeding the above-mentioned index continues, it can be predicted that the karyotypic abnormality of the adherent stem cells is increasing. When it is read that the karyotypic abnormality increases according to the index, the expression of the karyotypic abnormality in the adherent stem cells can be suppressed by appropriately changing the culture conditions (change in the seeding density, the culture medium, the growth factor, the serum, and the like) as needed. When the above-mentioned index is not satisfied, a cell population including adherent stem cells satisfying the above-mentioned index can be sorted by using, for example, a cell sorting technique. By reseeding and subculturing the adherent stem cells in the cell population, the expression of karyotype abnormality of the adherent stem cells can be suppressed. In the initial stage of the culture, the culture conditions (such as the seed density, the medium, the growth factor, and the serum) may be designed so as to satisfy the above-mentioned index at the final stage of the step, and the above-mentioned index may be satisfied at least at the final stage.
In the present invention, the quality of the donor itself and/or a biological sample collected from the donor can be evaluated by obtaining a cell population containing adherent stem cells from the donor, measuring the ratio of adherent stem cells positive to KCNAB1, and evaluating the ratio of adherent stem cells positive to KCNAB1 in the above cell population as an index, wherein the ratio is 85% or more. In the case where a cell population including adherent stem cells satisfying the above-described index is obtained (preferably, easily obtained), it can be confirmed that the quality of the donor and/or the biological sample collected from the donor is good. On the other hand, when the ratio of the cell population including the adherent stem cells exceeds the index, the quality of the biological sample collected from the donor is poor, and therefore, the occurrence of karyotypic abnormality of the adherent stem cells can be suppressed by appropriately changing the culture conditions (such as the inoculation density, the culture medium, the growth factor, and the serum). When the ratio of the cell population including the adherent stem cells exceeds the index, the karyotype abnormality of the adherent stem cells can be reduced by sorting the cell population including the adherent stem cells satisfying the index by, for example, a cell sorting technique, inoculating the adherent stem cells in the cell population, and culturing the cell population. Alternatively, by discarding a biological sample exceeding the above-mentioned index without culturing, the risk of obtaining a large amount of adherent stem cells of poor quality can be reduced. In the initial stage of the culture, the culture conditions (such as the seed density, the medium, the growth factor, and the serum) may be designed so as to satisfy the above-mentioned index at the final stage of the step, and the above-mentioned index may be satisfied at least at the final stage. In the case of confirming the quality of the biological sample collected from the donor, the method for preparing and treating the biological sample and the method for culturing the cell population are not particularly limited, and any method may be employed.
In the present invention, the optimal enzyme treatment conditions for the biological sample can be determined and/or predicted by measuring the ratio of adherent stem cells positive for KCNAB1 in a cell population obtained by subjecting the biological sample collected from a donor to an enzyme treatment and evaluating the cell population with the ratio of adherent stem cells positive for KCNAB1 in the cell population as an index of 85% or more. In the case where a cell population including adherent stem cells satisfying the above-described index is obtained (preferably, easily obtained), it is judged and/or predicted that an enzyme treatment method of a biological sample collected from a donor is appropriate. On the other hand, if the culture state whose value exceeds the above-mentioned index continues, it is judged and/or predicted that the method of treating the biological sample collected from the donor with the enzyme is not appropriate. In the case of determining and/or predicting the optimal enzyme treatment method, the method for preparing and treating a biological sample and the method for culturing a cell population are not particularly limited, and any method can be used.
The above-mentioned index is not particularly limited as long as it is measured at a desired timing, and includes: the present invention is not limited to the above-described examples, and the examples include the following examples, such as immediately after the isolation of cells from a biological sample, during the culture step, after purification in the culture step, immediately after n passages (n represents an integer of 1 or more), during the maintenance of culture, before cryopreservation, after thawing, or before the preparation becomes a pharmaceutical composition.
[5] Pharmaceutical composition
The cell population comprising adherent stem cells of the present invention can be used as a pharmaceutical composition. That is, according to the present invention, a pharmaceutical composition comprising the adherent stem cell-containing cell population of the present invention and a pharmaceutically acceptable vehicle can be provided. In addition, according to the present invention, a pharmaceutical composition comprising the cell population containing adherent stem cells of the present invention and other cells that can be administered can be provided.
The pharmaceutical composition of the present invention can be used as a cell therapeutic agent, for example, a therapeutic agent for intractable diseases.
The pharmaceutical composition of the present invention can be used as a pharmaceutical composition selected from the group consisting of immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, nervous system diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, connective tissue diseases, cerebral stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides, scleroderma, diseases caused by degeneration and/or inflammation of connective tissue such as cartilage, articular cartilage defects, meniscus injury, osteochondritis dissecans, aseptic necrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, ocular diseases, diseases related to angiogenesis, ischemic diseases, and inflammatory diseases, A therapeutic agent for a disease in coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart disease, trauma, epithelial injury, fibrosis, pulmonary disease, and cancer. The above-mentioned diseases can be treated by administering the pharmaceutical composition of the present invention to the treatment site in an amount capable of measuring the effect.
According to the present invention, a cell population comprising adherent stem cells of the present invention for use in a pharmaceutical composition can be provided.
According to the present invention, a cell population comprising adherent stem cells of the present invention for use in a cell therapeutic agent can be provided.
According to the present invention, there is provided a pharmaceutical composition for treating a disease selected from the group consisting of immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, nervous system diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, connective tissue diseases, stroke, cerebral infarction, intracerebral hematoma, cerebrovascular paralysis, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides, scleroderma, diseases caused by degeneration and/or inflammation of connective tissue such as cartilage, articular cartilage defects, meniscus injury, osteochondritis dissecans, aseptic necrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, ocular diseases, diseases related to angiogenesis, ischemic diseases, and inflammatory diseases, Cell populations comprising adherent stem cells of the invention for the treatment of diseases in coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart disease, trauma, epithelial injury, fibrosis, pulmonary disease, and cancer.
According to the present invention, a cell population comprising adherent stem cells of the present invention can be provided for administration to a patient or a subject for regeneration of cardiac muscle, generation of cardiac myocytes, angiogenesis, repair of blood vessels, or suppression of an immune response.
According to the present invention, there may be provided a method of transplanting cells to a patient or subject, and a method of treating a disease in a patient or subject, the method comprising: a step of administering to a patient or subject a therapeutically effective amount of a cell population comprising adherent stem cells of the invention.
According to the present invention, there may be provided the use of a cell population comprising adherent stem cells of the invention in the manufacture of a pharmaceutical composition.
According to the present invention, there may be provided the use of a cell population comprising adherent stem cells of the invention in the manufacture of a cell therapeutic.
According to the present invention, there is provided a cell population comprising adherent stem cells selected from the group consisting of immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, nervous system diseases, graft-versus-host disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, connective tissue diseases, stroke, cerebral infarction, intracerebral hematoma, cerebral palsy, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides, scleroderma, diseases caused by degeneration and/or inflammation of connective tissues such as cartilage, articular cartilage defects, meniscus injury, osteochondritis dissecans, aseptic necrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, ocular diseases, and the like, Use of a therapeutic agent for a disease associated with angiogenesis, ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart disease, trauma, epithelial injury, fibrosis, pulmonary disease, or cancer.
According to the present invention, there can be provided a use of the cell population containing adherent stem cells of the present invention for the manufacture of a therapeutic agent required for regeneration of cardiac muscle, generation of cardiac muscle cells, angiogenesis, repair of blood vessels, or suppression of immune response when administered to a patient or a subject.
The pharmaceutical composition of the present invention can be prepared by diluting a cell population comprising adherent stem cells with a pharmaceutically acceptable vehicle. The pharmaceutically acceptable vehicle is not particularly limited as long as it is a solution that can be administered to a patient or a subject. The pharmaceutically acceptable vehicle may be an infusion formulation, and may be exemplified by: examples of the "solution" include water for injection, physiological saline, 5% glucose solution, ringer's solution, lactated ringer's solution, acetate ringer's solution, bicarbonate ringer's solution, amino acid solution, starting solution (solution 1), dehydration supplement solution (solution 2), maintenance infusion solution (solution 3), postoperative recovery solution (solution 4), and Plasma-Lyte a (registered trademark).
The "patient or subject" in the present specification is typically a human, but may be other animals. Examples of other animals include: mammals such as dogs, cats, cows, horses, pigs, goats, sheep, monkeys (cynomolgus monkey, macaque, common marmoset, japanese monkey), ferrets, rabbits, rodents (mice, rats, gerbils, guinea pigs, hamsters), and birds such as chickens and quails, but the present invention is not limited thereto.
The term "treatment" as used herein includes, but is not limited to, a significant improvement in at least one of the life prognosis, the functional prognosis, the survival rate, the weight loss, anemia, diarrhea, dark stool, abdominal pain, fever, decreased appetite, malnutrition, vomiting, fatigue, eruption, inflammation, ulcer, erosion, fistula, stenosis, ileus, internal bleeding, rectal bleeding, spasm, pain, decreased liver function, decreased heart function, decreased lung function, and blood test items of a patient or a subject.
The pharmaceutical compositions of the present invention may comprise any ingredient used in the treatment of a patient or subject. Examples of the above-mentioned components include: salts (e.g., physiological saline, ringer's solution, BICANATE infusion), polysaccharides (e.g., hydroxyethyl starch (HES), dextran, etc.), proteins (e.g., albumin, etc.), dimethyl sulfoxide (DMSO), amino acids, medium components (e.g., components contained in RPMI1640 medium), etc., but are not limited thereto.
The pharmaceutical composition of the present invention may contain various additives for increasing storage stability, isotonicity, absorbability and/or viscosity, for example, emulsifiers, dispersants, buffers, preservatives, wetting agents, antioxidants, chelating agents, thickeners, gelling agents, pH adjusters and the like. Examples of the thickener include: HES, dextran, methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, and the like, but is not limited thereto. The concentration of the thickener varies with the thickener selected, and can be arbitrarily set within a range of concentrations that are safe and that achieve the desired viscosity when administered to a patient or subject.
The pharmaceutical composition of the present invention may further comprise 1 or more other drugs in addition to the adherent stem cells. Examples of the other drugs include: antibiotics, albumin preparations, vitamin preparations, anti-inflammatory agents, and the like, but are not limited thereto. As the above-mentioned anti-inflammatory agent, there may be mentioned: 5-aminosalicylic acid preparations, steroid preparations, immunosuppressants, biologics, etc., but are not limited thereto. Examples of the above 5-aminosalicylic acid preparation include: sulfasalazine, mesalamine, and the like, but are not limited thereto. Examples of the steroid preparation include: cortisone, prednisolone, methylprednisolone, and the like, but are not limited thereto. Examples of the immunosuppressive agent include: tacrolimus, cyclosporine, methotrexate, azathioprine, 6-mercaptopurine, and the like, but is not limited thereto. Examples of the biological agent include: infliximab (infliximab), adalimumab (adalimumab), uitlizumab (ustekinumab), secukinumab (secukinumab), epratuzumab (ixekizumab), brodalumab (brodalumab), tocilizumab (tocilizumab), vedolizumab (vedolizumab), felodib (filigonitinib), golimumab (golimumab), polyethylene glycol-conjugated certolizumab (certolizumab pegol), abasic (abatacept), etanercept (etanercept), and the like, but is not limited thereto.
In addition, the other drugs mentioned above may be other cells that can be administered. Examples of other cells to be administered include: blood-derived cells (leukocytes, erythrocytes, monocytes, etc.), vascular endothelial cells, vascular endothelial progenitor cells, pericytes, vascular wall cells, fibroblasts, skeletal myoblasts, epithelial cells, stromal cells, mature adipocytes, and the like, without particular limitation.
The pH of the pharmaceutical composition of the present invention may be a pH around neutral, for example, pH5.5 or more, 6.5 or more, or pH7.0 or more, or pH10.5 or less, pH9.5 or less, pH8.5 or less, or pH8.0 or less, but is not limited thereto.
When the pharmaceutical composition of the present invention is administered to a patient or a subject, the cell concentration is a cell concentration at which a therapeutic effect on a disease can be obtained as compared with a patient or a subject to which the pharmaceutical composition is not administered. The specific cell concentration can be determined appropriately according to the administration mode, the administration method, the purpose of use, and the age, weight, symptoms and the like of the patient or subject. The lower limit of the cell concentration of the pharmaceutical composition of the present invention is not particularly limited,for example, 1.0 × 1051.0 × 10 of more than one/mL61.2 × 10 of more than one/mL61.4 × 10 of more than one/mL61.6 × 10 of more than one/mL61.8 × 10 of more than one/mL62.0 × 10 of more than one/mL6More than 3.0 × 106More than one/mL, 4.0 × 1065.0 × 10 of more than one/mL6More than one/mL, 6.0 × 106More than one/mL, 7.0 × 106More than one/mL, 8.0 × 106More than 9.0 × 106More than 9.5 × 106More than one/mL, or 1.0 × 107The upper limit of the cell concentration of the pharmaceutical composition of the present invention is not particularly limited, but is, for example, 1.0 × 1010Less than 1/mL and 1.0 × 109Less than 8.0 × 10 pieces/mL8Less than 6.0 × 10 pieces/mL8Less than 4.0 × 10 pieces/mL82.0 × 10 below one/mL8Less than or equal to 1.0 × 108Less than one/mL.
The pharmaceutical composition of the present invention is preferably a liquid preparation, more preferably a liquid preparation for injection. As the liquid preparation for injection, for example, liquid preparations suitable for injection are known from international publication No. WO2011/043136, japanese patent application laid-open No. 2013-256510 and the like. The pharmaceutical composition of the present invention can be prepared into a liquid preparation for injection as described in the above-mentioned documents.
The liquid preparation may be a suspension of cells or a liquid preparation in which cells are dispersed in the liquid preparation. The form of the cells contained in the liquid preparation is not particularly limited, and may be, for example, single cells or cell aggregates.
In the case where the pharmaceutical composition of the present invention is a liquid preparation for injection, the lower limit of the cell concentration of the liquid preparation for injection is preferably 1.0 × 10 from the viewpoint of enhancing the therapeutic effect on diseases61.2 × 10 of more than one/mL61.4 × 10 of more than one/mL61.6 × 10 of more than one/mL61.8 × 10 of more than one/mL62.0 × 10 of more than one/mL6More than 3.0 × 106More than one/mL, 4.0 × 1065.0 × 10 of more than one/mL6More than one/mL, 6.0 × 106More than one/mL, 7.0 × 106More than one/mL, 8.0 × 106More than 9.0 × 106More than 9.5 × 106More than one/mL, or 1.0 × 107More than one cell per mL, and from the viewpoint of facilitating the preparation and administration of the liquid preparation for injection, the upper limit of the cell concentration of the liquid preparation for injection is preferably 1.0 × 109Less than 8.0 × 10 pieces/mL8Less than 6.0 × 10 pieces/mL8Less than 4.0 × 10 pieces/mL82.0 × 10 below one/mL8Less than or equal to 1.0 × 108Less than one/mL.
In addition, according to one embodiment of the present invention, the pharmaceutical composition of the present invention may be a preparation for transplantation. The preparation for transplantation is a solid or gel preparation, and examples of the solid preparation for transplantation include preparations for transplantation having a sheet-like structure or a granular structure. As a gel-structured transplant preparation, for example, a transplant preparation containing a gel obtained by binding isolated cells with a binder (e.g., fibrinogen) is known in international publication No. WO 2017/126549. In addition, according to one embodiment of the present invention, the pharmaceutical composition of the present invention may be a gel preparation in which cells and an arbitrary gel are mixed. As the gel preparation, for example, a cell therapeutic agent composed of an adherent stem cell-hydrogel composition is known in japanese patent publication No. 2017-529362. The pharmaceutical composition of the present invention can also be prepared into a gel preparation by using, for example, the method described in the above-mentioned document.
Further, as a preparation for transplantation having a sheet-like structure, for example, a cell sheet obtained by culturing using a temperature-responsive culture dish (for example, UpCell (registered trademark) (manufactured by CellSeed corporation)), a laminate of a sheet-like cell culture and fibrin gel, a cell-coated sheet obtained by coating a cell suspension on a sheet-like base material, and the like are known in international publication No. WO2006/080434, japanese patent application laid-open No. 2016-52272. The pharmaceutical composition of the present invention can be prepared into preparations for transplantation having various sheet-like structures by using, for example, the methods described in the above-mentioned documents.
The method of administering the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include: subcutaneous injection, intradermal injection, intramuscular injection, intralymphatic injection, intravenous injection, intraarterial injection, intraperitoneal injection, intrathoracic injection, direct local injection, direct sticking, or direct local transplantation, etc. According to one embodiment of the present invention, the liquid preparation for injection can be filled in a syringe and administered through an injection needle or a catheter into a vein, an artery, a myocardium, a joint cavity, a hepatic artery, a muscle, an epidural, a gum, a ventricle, a subcutaneous tissue, an intradermal tissue, an abdominal cavity, or a portal vein, but the present invention is not limited thereto. As for the method of administration of the pharmaceutical composition, for example, in Japanese patent laid-open No. 2015-61520, Onken JE, t al.American College of gastroenterology Conference 2006Las Vegas, NV, Abstract 121, Garcia-Olmo D, et al.Dis Colon Rectum 2005; 48:1416-23, etc. intravenous injection, intravenous drip, direct local injection, direct local transplantation, etc. are known. The pharmaceutical composition of the present invention can be administered by various methods described in the above documents.
The dose of the pharmaceutical composition of the present invention is an amount of cells which can obtain a therapeutic effect on a disease in comparison with a patient or a subject to which the pharmaceutical composition is not administered when administered to the patient or the subject, and the specific dose can be appropriately determined depending on the administration form, the administration method, the purpose of use, the age, weight, symptoms and the like of the patient or the subject, and the dose of 1 dose of human adherent stem cells is not particularly limited, and is, for example, 1 × 1041 × 10 above body weight/kg55 × 10 above per kg body weight51 × 10 above body weight/kg62 × 10 of more than one/kg body weight64 × 10 above body weight/kg66 × 10/kg body weight of the human body6More than or equal to 8 × 10 per kg body weight6The dose per kg body weight of the human adherent stem cells is not particularly limited to 1 dose, and is, for example, 1 × 1012Less than 1/kg body weight, 1 × 1011Less than 1/kg body weight, 1 × 1010Less than 1/kg body weight, 1 × 109Less than 5/kg body weight, 5 × 108Less than 1/kg body weight, 1 × 108Less than one/kg body weight、8×107Less than 6/kg body weight, 6 × 107Less than 4 × 10 per kg body weight7Less than 2/kg body weight, or 2 × 107Less than one/kg body weight.
In the case where the pharmaceutical composition of the present invention is a liquid preparation for injection, the 1 dose of the liquid preparation for injection to human adherent stem cells is preferably 1 × 10 from the viewpoint of enhancing the therapeutic effect on diseases 55 × 10 above per kg body weight 51 × 10 above body weight/kg 62 × 10 of more than one/kg body weight 64 × 10 above body weight/kg66 × 10/kg body weight of the human body6More than or equal to 8 × 10 per kg body weight6More than one per kg body weight, and from the viewpoint of ease of preparation and administration of the liquid preparation for injection, 1 dose of the liquid preparation for injection to human adherent stem cells is preferably 1 × 109Less than 5/kg body weight, 5 × 108Less than 1/kg body weight, 1 × 108Less than 8/kg body weight, 8 × 107Less than 6/kg body weight, 6 × 107Less than 4 × 10 per kg body weight7Less than 2/kg body weight, or 2 × 107Less than one/kg body weight.
The frequency of administration of the pharmaceutical composition of the present invention is a frequency at which a therapeutic effect on a disease can be obtained when the pharmaceutical composition is administered to a patient or a subject. The specific administration frequency can be determined appropriately according to the administration form, administration method, purpose of use, and age, body weight, symptoms, and the like of the patient or subject, and is, for example, 1 time for 4 weeks, 1 time for 3 weeks, 1 time for 2 weeks, 1 time for 1 week, 2 times for 1 week, 3 times for 1 week, 4 times for 1 week, 5 times for 1 week, 6 times for 1 week, or 7 times for 1 week.
The period of administration of the pharmaceutical composition of the present invention is a period in which a therapeutic effect on a disease can be obtained when the pharmaceutical composition is administered to a patient or a subject. The specific administration period may be appropriately determined depending on the administration form, administration method, purpose of use, and age, body weight, symptoms, and the like of the patient or subject, and is, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.
The timing of administering the pharmaceutical composition of the present invention to a patient or a subject is not particularly limited, and examples thereof include: immediately after onset, within n days from onset (n represents an integer of 1 or more), immediately after diagnosis, within n days from diagnosis (n represents an integer of 1 or more), before remission, during remission, after remission, before relapse, during relapse, after relapse, and the like.
The pharmaceutical composition of the present invention may be stored in a frozen state until just before use. The temperature for cryopreservation is preferably-30 deg.C or lower, -40 deg.C or lower, -50 deg.C or lower, -80 deg.C or lower, -90 deg.C or lower, -100 deg.C or lower, -150 deg.C or lower, -180 deg.C or lower, or-196 deg.C or lower (liquid nitrogen temperature). When the pharmaceutical composition of the present invention is administered to a patient or a subject, it can be used by rapid thawing at 37 ℃.
Other examples of diseases and the like that may be treated with a population of cells comprising adherent stem cells in a patient or subject, other specific examples of such diseases and the like, and specific steps of treatment may be found in Hare et al, j.am.coll.cardiol.,2009December 8; 54(24) 2277-2286, Honmou et al, Brain 2011: 134; 1790-; 95:1827-1833, Japanese patent No. 590577, Japanese patent application No. 2010-518096, Japanese patent application No. 2012-509087, Japanese patent application No. 2014-501249, Japanese patent application No. 2013-256515, Japanese patent application No. 2014-185173, Japanese patent application No. 2010-535715, Japanese patent application No. 2015-038059, Japanese patent application No. 2015-110659, japanese laid-open patent publication Nos. 2006-521121, 2009-542727, 2014-224117, 2015-061862, 2002-511094, 2004-507454, 2010-505764, 2011-514901, 2013-064003, 2015-795, and the like.
The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.
Examples
< comparative example 1 >
In comparative example 1 and example 1 shown below, an index for obtaining a cell population including adherent stem cells having high karyotype stability was examined.
(step 1-1: collection of amnion)
Fetal membranes and placentas were aseptically collected as fetal appendages from pregnant women who had received informed consent for elective caesarean delivery cases. The obtained fetal membranes and placenta were placed in a sterilized pot containing physiological saline, and the amnion was manually peeled off from the cut ends of the fetal membranes. The amniotic membrane was washed with Hank's balanced salt solution (Ca/Mg free) to remove adherent blood and blood clots.
(step 1-2: enzyme treatment of amnion and recovery of amnion-adherent stem cells)
The amnion containing the epithelial cell layer and the adherent stem cell layer was immersed in a Hank's balanced salt solution (containing Ca/Mg) containing 240PU/mL collagenase and 200PU/mL Dispase I, and subjected to an enzymatic treatment by shaking at 37 ℃ for 90 minutes at 50 rpm. The enzyme-treated solution was filtered through a nylon mesh having a mesh size of 95 μm to remove undigested amnion, thereby recovering a cell suspension containing amnion-adherent stem cells.
(step 1-3: culture of amnion adherent Stem cells)
The cell population containing the amnion-adherent stem cells obtained in the above-described "enzymatic treatment of amnion and recovery of amnion-adherent stem cells" was treated with 6,000cells/cm2The cell density of (a) was inoculated in a culture vessel CellSTACK (registered trademark) (manufactured by Corning Co., Ltd.), and the cell was cultured in α MEM (alpha Modification of Minimum Essential Medium eagle) containing 10% of bovine fetal serum (FBS) and 10ng/mL of basic fibroblast growth factor (bFGF) in final concentration until near confluency (subonfluence). then, the 0 th passage cell was detached using a TrypLE Select, and the 1/5 amount of cells were inoculated in the same scale of the CellSTACK (registered trademark) as the previous culture, whereby subculture was performed, Medium replacement was performed at a frequency of 2 to 4 days and 1 time, the 1 st passage cell was detached using a TrypLE Select at the time of reaching near confluency, and RPMI1640 was added so that the cell concentration was 2 × 107cells/mL. To this was added an equal amount of CP-1 (registered trademark) solution (CP-1 (Note)Trade mark) to 25% human serum albumin at a ratio of 34: 16), transferred to a cryopreservation vial at 1mL per portion, slowly frozen to-80 ℃, and then cryopreserved in liquid nitrogen for 1 day. Then, the mixture is thawed to about 15,000 to 18,000cells/cm2The density of (1) was determined by inoculating cells of the 2 nd passage into CellSTACK (registered trademark), culturing the cells in α MEM (alpha Modification of Minimum Essential Medium eagle) containing 10% of bovine fetal serum (FBS) and 10ng/mL of basic fibroblast growth factor (bFGF) in final concentration until they reach near confluence, then removing the cells of the 2 nd passage by using TrypLE Select, inoculating 1/5 amount of cells into CellSTACK (registered trademark) of the same scale as the previous culture, thereby performing subculture, changing the Medium at a frequency of 1 time for 2-4 days, removing the cells of the 3 rd passage by using TrypLE Select at the time of reaching near confluence, and adding RPMI1640 to obtain a cell concentration of 4 × 106cells/mL. To this, an equal amount of CP-1 (registered trademark) solution (a mixed solution of CP-1 (registered trademark) and 25% human serum albumin in a ratio of 34: 16) was added, transferred to a cryopreservation vial in an amount of 1mL each, slowly frozen to-80 ℃, and then cryopreserved in liquid nitrogen for 1 day. Then, thawing was performed at about 6,000cells/cm2The 4 th passage cells were inoculated into CellSTACK (registered trademark), and adherent-cultured in α MEM (alpha Modification of Minimum essential Medium eagle) containing 10% of bovine fetal serum (FBS) and 10ng/mL of basic fibroblast growth factor (bFGF) in final concentration to near confluence, then the 4 th passage cells were detached using TrypLE Select, and 1/5 amount of cells were inoculated into CellSTACK (registered trademark) of the same scale as the previous culture, whereby the passage culture was performed, the medium replacement was performed at a frequency of 1 time for 2 to 4 days, the 5 th passage cells were detached using TrypLE Select at the time of reaching near confluence, and RPMI1640 was added so that the cell concentration was 4 × 106cells/mL. To this, an equal amount of CP-1 (registered trademark) solution (a mixed solution of CP-1 (registered trademark) and 25% human serum albumin in a ratio of 34: 16) was added, transferred to a cryopreservation vial in an amount of 1mL each, slowly frozen to-80 ℃, and then cryopreserved in liquid nitrogen for 1 day.
(Steps 1-4: antigen analysis of amnion adherent Stem cells)
Amnion adherent stem cells at passage 5 cultured by the above-described culture method were analyzed for various surface antigens (known as MSC markers, positive rate of CD73, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD34, and negative rate of CD326) using a flow cytometer. As a result, the positive rates for CD73, CD90 and CD105 were all 90% or more (specifically, CD 73: 100%, CD 90: 97%, CD 105: 100%), and the positive rate for CD166 was all 30% or more (specifically, CD 166: 98%). The negative rates for CD45, CD34, and CD326 were all 95% or more (specifically, CD 45: 100%, CD 34: 100%, and CD 326: 100%). From the above results, it was confirmed that the cells cultured by the above culture method were adherent stem cells.
The amnion-adherent stem cells at passage 3 and passage 5 cultured by the above-described culture method were fixed and membrane permeabilized, and the percentage of cells positive to KCNAB1 antigen was analyzed by flow cytometry. As a result, the number of passage was less than 85% (3 rd passage: 78%, 5 th passage: 81%).
In the present assay, as an antibody for Isotype Control, FITC Mouse IgG1,. kappa.Isotype Control (BD Co./model: 550616), PE Mouse IgG1,. kappa.Isotype Control (BD Co./model: 555749), as an antibody against CD73 antigen, FITC Mouse Anti-Human CD73(BD Co./model: 561254), as an antibody against CD90 antigen, FITC Mouse Anti-Human CD90(BD Co./model: 555595), as an antibody against CD105 antigen, Anellandies FITC Conjugate (BioLegend Co./model: 323203), as an antibody against CD166 antigen, PE Mouse-Anti-CD 166(BD Co./model: 559263), as an antibody against CD45, Mouse C-Humti-CD 8536, as an antibody against CD73 antigen, PE Mouse Anti-Human CD34(BD Co., Ltd.: 555822) was used, and FITC Mouse Anti-Human EpCAM (BD Male) was used as an antibody against CD326 antigenDepartment/model: 347197), as an antibody against KCNAB1 antigen, KCNAB1 monoclonal antibody (FITC) (Abnova corporation/model: MAB 11866). Surface antigen analysis and antigen analysis Using BD Accuri of Becton, Dickinson and Company (BD)TMC6 Flow Cytometer, assay conditions for analyzing cell number: 5,000cells, flow rate setting: slow (14. mu.L/min). The ratio of positive cells to each antigen was calculated by the following procedure.
(1) The measurement results are plotted in a dot diagram with the side scattered light (SSC) on the vertical axis and the FSC (forward scattered light) on the horizontal axis.
(2) In the dot diagram, the entire region (gate) in which the fluorescence intensity of all cells measured with the antibody for isotype control is higher than 0.5% was selected.
(3) The ratio of cells contained in the gate selected in (2) among all the cells measured with the antibody corresponding to the antigen marker was calculated.
(step 1-5: analysis of Gene expression of amnion-adherent Stem cells)
The amnion-adherent stem cells of the 5 th passage cultured by the above-mentioned culture method were subjected to microarray-based gene (KCNAB1 gene, SULT1E1 gene, MN1 gene, RARRES2 gene) expression analysis by RIKENGENESIS.
Microarray analysis was performed according to the following steps (1) to (4). The following steps (2) to (4) are carried out by RIKEN GENESIS.
(1) The cryopreserved cell population was thawed and recovered by centrifugation. The recovered cell population was washed with Phosphate Buffered Saline (PBS), and the cells were recovered by centrifugation. Then, total RNA was extracted and purified using Rneasy Plus Mini kit (QIAGEN).
(2) cDNA was synthesized from 100ng of total RNA by reverse transcription reaction, and transcribed from cDNA to cRNA by in vitro transcription, and biotin-labeled (using 3' IVT PLUS Reagent Kit).
(3) 10.0. mu.g of labeled cRNA was added to the hybridization buffer, and hybridization was performed for 16 hours on a Human Genegenome U133A 2.0.0 Array (manufactured by Affymetrix). After washing with GeneChip fluids Station 450 (manufactured by Affymetrix), phycoerythrin staining was performed, and then scanning was performed with GeneChip Scanner 30007G (manufactured by Affymetrix), image analysis was performed with AGCC (Affymetrix GeneChip Command Console software (manufactured by Affymetrix), and numerical analysis was performed with Affymetrix Expression Console (manufactured by Affymetrix).
(4) The numerical data file was analyzed using analysis software GeneSpring GX (Agilent Technologies, Inc.).
The expression level of each gene was determined as a relative expression amount with respect to the expression level of the SDHA gene. As a result, the KCNAB1 gene: 0.03, SULT1E1 gene: 0.05, MN1 gene: 0.69, RARRES2 gene: 0.41.
(Steps 1-6: karyotyping of amnion-adherent stem cells)
With respect to the amnion-adherent stem cells cultured by the above-mentioned culture method at 3 rd and 5 th passages, karyotype analysis by the G-band method was performed by Japanese Gene research institute. Cells frozen at passages 3 and 5 were thawed at about 8,000cells/cm2Is inoculated into each of 2T 25 flasks, 5mL of α MEM containing 10% of bovine fetal serum (FBS) in final concentration and 10ng/mL of basic fibroblast growth factor (bFGF) is added thereto, and then the T25 flask is filled with α MEM containing 10% of bovine fetal serum (FBS) in final concentration and 10ng/mL of basic fibroblast growth factor (bFGF) at room temperature, transported to japan gene research institute, japan, inc., ltd, collects arbitrary 20 cells, takes out chromosomes therefrom, identifies each chromosome based on a characteristic band pattern in chromosomes detected by banding technique, analyzes whether there is a karyotype abnormality such as aneuploidy and translocation, and, as a result, a karyotype abnormality is confirmed in any cell of 3 rd and 5 th passages (specifically, 1 of 20 cells is confirmed in 3 rd passage, 2 nd passage 2 th chromosome abnormality is confirmed in 2 nd passage 2 th passage, and 82 th passage 2 nd passage 2 th passage of chromosome is confirmed in 3 rd passage 2 nd passage, and the same number of chromosomes is increased in 365 th passage 2 nd passage (82), and the same number of chromosomes is confirmed in 3 rd passage 2 nd passage)Trisomy in chromosome 2 was identified.
< example 1 >
(step 2-1: collection of amnion)
Amnion was obtained by the same method as in comparative example 1.
(step 2-2: enzyme treatment of amnion and recovery of amnion-adherent stem cells)
A cell population comprising amnion-adherent stem cells was obtained by the same method as in comparative example 1.
(step 2-3: culture of amnion adherent Stem cells)
The cell population containing the amnion-adherent stem cells obtained in the above-described "enzymatic treatment of amnion and recovery of amnion-adherent stem cells" was treated with 6,000cells/cm2The cell density of (2) was inoculated to CellSTACK (registered trademark), and adherent culture was performed in α MEM containing human platelet lysate (hPL) at a final concentration of 5% until the cell density reached near confluence, and then, the 0 th passage cell was detached using TrypLE Select, and 1/5 number of cells were inoculated to CellSTACK (registered trademark) on the same scale as the previous culture, thereby subculture was performed, and medium replacement was performed at a frequency of 1 time in 2 to 4 days, and the 1 st passage cell was detached using TrypLE Select at the time of reaching near confluence, and RPMI1640 was added so that the cell concentration was 2 × 107cells/mL. To this, an equal amount of CP-1 (registered trademark) solution (a mixed solution of CP-1 (registered trademark) and 25% human serum albumin in a ratio of 34: 16) was added, transferred to a cryopreservation vial in an amount of 1mL each, slowly frozen to-80 ℃, and then cryopreserved in liquid nitrogen for 1 day. Then, the mixture is thawed to about 15,000 to 18,000cells/cm2The density of (2) passage cells were inoculated into CellSTACK (registered trademark), and adherent culture was performed in α MEM containing human platelet lysate (hPL) at a final concentration of 5% to near confluence, and then the 2 nd passage cells were detached using TrypLE Select, and 1/5 amount of cells were inoculated into CellSTACK (registered trademark) on the same scale as the previous culture, whereby subculture was performed, medium replacement was performed at a frequency of 2 to 4 days and 1 time, the 3 rd passage cells were detached using TrypLE Select at the time of reaching near confluence, and RPMI was added so that the cell concentration was 16404×106cells/mL. To this, an equal amount of CP-1 (registered trademark) solution (a mixed solution of CP-1 (registered trademark) and 25% human serum albumin in a ratio of 34: 16) was added, transferred to a cryopreservation vial in an amount of 1mL each, slowly frozen to-80 ℃, and then cryopreserved in liquid nitrogen for 1 day. Then, thawing was performed at about 6,000cells/cm2The density of (2) was determined by inoculating the cells of the 4 th passage in CellSTACK (registered trademark), culturing the cells adherent to α MEM containing human platelet lysate (hPL) at a final concentration of 5% until they were nearly confluent, then detaching the cells of the 4 th passage using TrypLE Select, inoculating 1/5 amount of the cells in CellSTACK (registered trademark) on the same scale as the previous culture, subculturing the cells, replacing the medium at a frequency of 2 to 4 days and 1 time, detaching the cells of the 5 th passage using TrypLE Select at the time of reaching nearly confluent, and adding RPMI1640 so that the cell concentration was 4 × 106cells/mL. To this, an equal amount of CP-1 (registered trademark) solution (a mixed solution of CP-1 (registered trademark) and 25% human serum albumin in a ratio of 34: 16) was added, transferred to a cryopreservation vial in an amount of 1mL each, slowly frozen to-80 ℃, and then cryopreserved in liquid nitrogen. After passage 6, all at approximately 6,000cells/cm2The cells were seeded on CellSTACK (registered trademark), and adherent culture was performed in α MEM containing human platelet lysate (hPL) at a final concentration of 5% to near confluence, followed by dissection using TrypLE Select, and repeated subculture was performed until passage 9.
(step 2-4: antigen analysis of amnion adherent Stem cells)
The amnion-adherent stem cells at passage 5 cultured by the above-described culture method were analyzed for various surface antigens (positive rate of CD73, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD34, and negative rate of CD326) using a flow cytometer. As a result, the positive rates of CD73, CD90 and CD105 were all 50% or more (specifically, CD 73: 99%, CD 90: 100% and CD 105: 100%). The positive rate for CD166 was 30% or more (specifically, CD 166: 100%). The negative rates for CD45, CD34, and CD326 were all 95% or more (specifically, CD 45: 100%, CD 34: 100%, and CD 326: 100%). From the above results, it was confirmed that the cells cultured by the above culture method were adherent stem cells.
The percentage of amnion-adherent stem cells at passages 3 and 5 cultured by the above-described culture method that were positive for KCNAB1 antigen was analyzed by flow cytometry. As a result, the number of passage was 85% or more (91% in the 3 rd passage and 90% in the 5 th passage). From this, it was found that the amnion adherent stem cells at passage 5 of example 1 satisfy the condition that the ratio of adherent stem cells positive to KCNAB1 is 85% or more.
The method and reagents for this measurement were the same as in comparative example 1.
(step 2-5: analysis of Gene expression of amnion-adherent Stem cells)
The amnion-adherent stem cells of the 5 th passage cultured by the above-mentioned culture method were subjected to microarray-based gene (KCNAB1 gene, SULT1E1 gene, MN1 gene, RARRES2 gene) expression analysis by RIKENGENESIS.
Microarray analysis was performed in the same manner as in comparative example 1. The expression level of each gene was determined as a relative expression amount with respect to the expression level of the SDHA gene. As a result, the KCNAB1 gene: 0.44, SULT1E1 gene: 0.68, MN1 gene: 1.77, RARES 2 gene: 0.003.
(step 2-6: karyotyping of amnion-adherent stem cells)
With respect to the amnion-adherent stem cells cultured by the above-mentioned culture method at 3 rd and 5 th passages, karyotype analysis by the G-band method was performed by Japanese Gene research institute. Cells frozen at passages 3 and 5 were thawed at about 8,000cells/cm2Was inoculated into 2 respective T25 flasks, and 5mL of α MEM containing 5% of human platelet lysate (hPL) at the final concentration was added thereto, and the resulting mixture was cultured for 24 hours, and then the T25 flask was filled with α MEM containing 5% of human platelet lysate (hPL) at the final concentration, and the resulting mixture was transported to the japan gene research institute of japan (fairy, city, japan) at room temperatureChromosomes were extracted from any 20 cells, and the chromosomes were identified based on characteristic band patterns in chromosomes detected by the banding technique, and the presence or absence of karyotypic abnormalities such as heteroploidy and translocation was analyzed. As a result, all cells at passages 3 and 5 maintained normal karyotype.
The positive rate and karyotype analysis results of KCNAB1 in comparative example 1 and example 1 are summarized in table 1.
[ Table 1]
KCNAB1 positive rate and karyotype analysis results described in examples and comparative examples
Figure BDA0002557821570000401
From the above results, it was found that the cell population having a positive rate of KCNAB1 of 85% or more retained the normal karyotype. Further, it was found that the condition that the positive rate of KCNAB1 was 85% or more was effective as an index for obtaining a cell population containing adherent stem cells that retained the normal karyotype. That is, according to the present invention, a cell population including adherent stem cells that retain a normal karyotype can be obtained by using as an indicator a condition that the positive rate of KCNAB1 is 85% or more. Thus, a safe cell preparation suitable for clinical use can be prepared.
Further, if the condition that the positive rate of KCNAB1 is 85% or more is used as an index, the karyotype stability of the biological sample can be evaluated (judged and/or predicted) without performing the karyotype analysis which usually requires an evaluation period of about 1 month.
Furthermore, according to the present invention, it is possible to monitor the karyotypic stability (presence or absence of karyotypic abnormality in adherent stem cells) of a biological sample over time, simply and in a short time, by using as an index that the positive rate of KCNAB1 in a cell population containing adherent stem cells is 85% or more. This can reduce the cost required for quality evaluation of a biological sample, shorten the time required for quality evaluation, and reduce the production cost of a cell preparation.
< example 2 >
Fetal membranes and placentas as fetal appendages were aseptically collected from pregnant women who obtained informed consent cases of elective caesarean section of 2 donors (#1, #2) different from the donors in comparative example 1 and example 1, and the collected material was subjected to "procedure 1-1: collection of amnion "," procedure 1-2: and (3) treating each fetal appendage by amnion enzyme treatment and amnion adherent stem cell recovery to obtain amnion adherent stem cells.
Using "example 1: culture of amnion adherent stem cells "the method cultures a cell population containing amnion adherent stem cells obtained from a #1 or #2 donor to obtain a cell population at passage 5.
The amnion adherent stem cells cultured by the above-described culture method may be prepared in the same manner as in "steps 1 to 4: antigen analysis of amnion adherent stem cells "the ratio of cells positive to KCNAB1 was also analyzed using a flow cytometer. The positive rates of KCNAB1 were 85% or more (# 1: 94%), and less than 85% (49% in particular) for # 2. The amnion-adherent stem cells cultured by the above-described culture method may be prepared in the same manner as in "steps 1 to 6: karyotyping of amnion adherent stem cells "karyotype was analyzed similarly. As a result of karyotyping analysis, the normal karyotype was maintained in all the cells of #1, but karyotypic abnormality was confirmed in #2 (specifically, karyotypic abnormality was confirmed in 1 of 20 cells, and structural abnormality of 13 th chromosome (add (13) (p 11.2))).
The positive rate and karyotype analysis results for KCNAB1 in example 2 are summarized in table 2.
[ Table 2]
KCNAB1 Positive Rate and karyotype analysis results described in example 2
Figure BDA0002557821570000411
< example 3>
In example 3 shown below, amnion-adherent stem cells having different donors, enzyme treatment conditions, and culture conditions were obtained as compared with comparative example 1, and example 2.
Fetal membranes and placentas as fetal appendages were aseptically collected from pregnant women who obtained informed consent cases of elective caesarean section of 3 different donors from those in comparative example 1, example 1 and example 2 as donors (#3 to # 5).
(step 3-1: collection of amnion)
Amnion was obtained by the same method as in comparative example 1.
(step 3-2: enzyme treatment of amnion and recovery of amnion-adherent stem cells)
Amnion containing epithelial cell layer and adherent stem cell layer was immersed in Hank's balanced salt solution (containing Ca/Mg) containing 480PU/mL collagenase and 400PU/mL Dispase I, and subjected to enzyme treatment by shaking at 37 ℃ for 90 minutes at 50 rpm. The enzyme-treated solution was filtered through a nylon mesh having a mesh size of 95 μm to remove undigested amnion, thereby recovering a cell suspension containing amnion-adherent stem cells.
(step 3-3: culture of amnion adherent Stem cells)
The cell population containing the amnion-adherent stem cells obtained in the above-described "enzymatic treatment of amnion and recovery of amnion-adherent stem cells" was treated with 1,000cells/cm2The cell density of (2) was inoculated to CellSTACK (registered trademark), and adherent culture was performed in α MEM containing human platelet lysate (hPL) at a final concentration of 5% until the cell density reached near confluence, medium replacement was performed at a frequency of 1 time for 3 to 5 days, and then the 0 th passage cells were detached using TrypLE Select, and the cell concentration was added at 2 × 107cells/mL physiological saline. To this, an equal amount of CP-1 (registered trademark) solution (a mixed solution of CP-1 (registered trademark) and 25% human serum albumin in a ratio of 34: 16) was added, transferred to a cryopreservation vial in an amount of 1mL each, slowly frozen to-80 ℃, and then cryopreserved in liquid nitrogen for 1 day. Then, thawing was performed at about 1,000cells/cm2(ii) cell of passage 1 was seeded on CellSTACK (registered trademark), and adherent culture was performed in α MEM containing human platelet lysate (hPL) at a final concentration of 5% for 5 days to near confluence, and then, the cell of passage 1 was detached using TrypLE Select, and physiological saline was added so that the cell concentration was at a level of2×107cells/mL. To this, an equal amount of CP-1 (registered trademark) solution (a mixed solution of CP-1 (registered trademark) and 25% human serum albumin in a ratio of 34: 16) was added, transferred to a cryopreservation vial in an amount of 1mL each, slowly frozen to-80 ℃, and then cryopreserved in liquid nitrogen for 1 day. Then, thawing was performed at about 1,000cells/cm2(ii) cell of the 2 nd passage was seeded in CellSTACK (registered trademark), and adherent culture was performed in α MEM containing human platelet lysate (hPL) at a final concentration of 5% for 5 days to near confluence, and then, the cell of the 2 nd passage was detached using TrypLE Select, and physiological saline was added so that the cell concentration was 4 × 106cells/mL. To this, an equal amount of CP-1 (registered trademark) solution (a mixed solution of CP-1 (registered trademark) and 25% human serum albumin in a ratio of 34: 16) was added, transferred to a cryopreservation vial in an amount of 1mL each, slowly frozen to-80 ℃, and then cryopreserved in liquid nitrogen.
(step 3-4: antigen analysis of amnion adherent Stem cells)
The amnion-adherent stem cells of the 2 nd passage cultured by the above-described culture method were analyzed for various surface antigens (positive rate of CD73, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD34, and negative rate of CD326) using a flow cytometer. As a result, the positive rates of CD73, CD90, and CD105 were all 50% or more (specifically, all of #3 to #5 were 100%). The positive rates of CD166 were all 30% or more (specifically, # 3: 99%, # 4: 100%, # 5: 99%). The negative rates for CD45, CD34, and CD326 were all 95% or more (specifically, # 3: 99%, # 4: 100%, # 5: 100%). From the above results, it was confirmed that the cells cultured by the above culture method were adherent stem cells.
The ratio of cells positive to KCNAB1 antigen was analyzed by flow cytometry on the amnion-adherent stem cells at passage 2 cultured by the above-described culture method. As a result, 85% or more of all donors were observed (# 3: 98.2%, # 4: 99.9%, # 5: 99.7%). From this, it was found that the amnion adherent stem cells at passage 2 of example 3 satisfy the condition that the ratio of adherent stem cells positive to KCNAB1 is 85% or more for all donors #3 to # 5. The method and reagents for this measurement were the same as in comparative example 1.
(step 3-5: analysis of Gene expression of amnion-adherent Stem cells)
The amnion-adherent stem cells of the 2 nd passage cultured by the above-mentioned culture method were subjected to microarray-based gene (KCNAB1 gene, SULT1E1 gene, MN1 gene, RARRES2 gene) expression analysis by RIKENGENESIS.
Microarray analysis was performed in the same manner as in comparative example 1. The expression level of each gene was determined as a relative expression amount with respect to the expression level of the SDHA gene. As a result, #3 was the KCNAB1 gene: 0.15, SULT1E1 gene: 0.36, MN1 gene: 0.81, RARRES2 gene: 0.01. #4 is the KCNAB1 gene: 0.07, SULT1E1 gene: 0.16, MN1 gene: 0.75, RARRES2 gene: 0.001. #5 is the KCNAB1 gene: 0.11, SULT1E1 gene: 0.27, MN1 gene: 1.38, RARES 2 gene: 0.001.
furthermore, the expression analysis of the gene (KCNAB1 gene, SULT1E1 gene, MN1 gene, RARRES2 gene) by quantitative PCR was performed on the amnion-adherent stem cells of passage 2 cultured by the above-described culture method. Quantitative PCR-based gene expression analysis was performed according to the following steps (1) to (4).
(1) The cryopreserved cell population was thawed and recovered by centrifugation. The recovered cell population was washed with Phosphate Buffered Saline (PBS), and the cells were recovered by centrifugation. Then, total RNA was extracted and purified using Rneasy Plus Mini kit (QIAGEN).
(2) To the purified total RNA, ReverTra Ace qPCR RT Master Mix (manufactured by Toyo Kagaku Co., Ltd.) was added, and cDNA was synthesized by reverse transcription using the total RNA as a template.
(3) The synthesized cDNA was mixed with Taqman Fast Advanced Master Mix (Applied Biosystems) and primers (Taqman Gene Expression Assay, Thermo Fisher, primer Assay ID of SDHA: Hs00188166_ m1, primer Assay ID of KCNAB 1: Hs00185764_ m1, primer Assay ID of SULT1E 1: Hs00960938_ m1, primer Assay ID of MN 1: Hs00159202_ m1, primer Assay ID of RARES 2: Hs00414615_ m1) and injected into a 96-well plate to perform quantitative PCR.
(4) The delta Ct value of each sample relative to SDHA was analyzed by StepOneNus Real-Time PCR System (Applied Biosystems), and the relative expression level (2^ (-delta Ct)) of each gene relative to the expression level of SDHA gene in each cell was calculated. As a result, #3 was the KCNAB1 gene: 0.77, SULT1E1 gene: 0.37, MN1 gene: 1.1, RARES 2 gene: 0.0026. #4 is the KCNAB1 gene: 0.72, SULT1E1 gene: 0.13, MN1 gene: 1.8, RARES 2 gene: 0.0029. #5 is the KCNAB1 gene: 4.4, SULT1E1 Gene: 0.50, MN1 gene: 5.9, RARES 2 gene: 0.0033.
(step 3-6: karyotyping of amnion-adherent stem cells)
With respect to the amnion-adherent stem cells of the 2 nd passage cultured by the above-mentioned culture method, karyotype analysis by the G-band method was performed by Japanese institute of genetics, Inc. The cells frozen at passage 2 were thawed at about 8,000cells/cm2Was inoculated into each of 2T 25 flasks, and cultured for 24 hours with 5mL of α MEM added thereto, the α MEM containing 5% of human platelet lysate (hPL) at the final concentration, then the T25 flask was filled with α MEM containing 5% of human platelet lysate (hPL) at the final concentration, transported to japan gene research institute, inc.
From the above results, it was found that the cell population having a positive rate of KCNAB1 of 85% or more retained the normal karyotype. It was also shown that even when amnion adherent stem cells were collected/cultured from amnion by a different method, the quality of the donor itself and the biological sample collected from the donor could be evaluated if the positive rate of KCNAB1 was 85% or more. That is, according to the present invention, a biological sample having a high content of amnion adherent stem cells free from karyotype abnormality can be selected (donor screening) by using as an indicator that the positive rate of KCNAB1 is 85% or more. Further, the enzyme treatment conditions and the culture conditions can be optimized (improvement of the enzyme treatment conditions and the culture conditions) by using as an index that the positive rate of KCNAB1 is 85% or more. This can shorten the time required for quality evaluation, and can reduce the time required for improvement of the production method such as enzyme treatment conditions and culture conditions.
< example 4 >
A part of the amnion-adherent stem cells obtained in example 1 was used for preparation of a pharmaceutical composition, and amnion-adherent stem cells 2.0 × 10-containing cells were prepared86.8mL of CP-1 solution (registered trademark), 3.2mL of 25% human serum albumin solution, and 10mL of RPMI1640 medium. The pharmaceutical composition is sealed in a freezing bag and stored in a frozen state. The pharmaceutical composition may be thawed and administered to a patient at the time of use.
< reference example >
(step 4-1: culture of mesenchymal Stem cells derived from bone marrow)
3 donor portions (#6 to #8) of human bone marrow-derived mesenchymal stem cells (hMSC mesenchymal stem cells, manufactured by Lonza) were purchased and thawed at 6,000cells/cm2The cell density of (2) was inoculated on a culture dish of phi 15cm, and adherent culture was carried out in a special medium manufactured by Lonza until the cell density reached nearly confluency, the medium replacement was carried out at a frequency of 1 time for 3 to 5 days, then cells were detached using trypLESelect, and physiological saline was added so that the cell concentration was 2 × 106cells/mL. To this, an equal amount of CP-1 (registered trademark) solution (a mixed solution of CP-1 (registered trademark) and 25% human serum albumin in a ratio of 34: 16) was added, transferred to a cryopreservation vial in an amount of 1mL each, slowly frozen to-80 ℃, and then cryopreserved in liquid nitrogen.
(step 4-2: antigen analysis of mesenchymal Stem cells derived from bone marrow)
The bone marrow-derived mesenchymal stem cells cultured by the above-described culture method were analyzed for various surface antigens (positive rate of CD73, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD34, and negative rate of CD326) by using a flow cytometer. As a result, the positive rates of CD73, CD90, and CD105 were all 50% or more. The positive rate of CD166 is more than 30%. CD45, CD34 and CD326 were all less than 5%.
In addition, the ratio of cells positive to KCNAB1 antigen was analyzed using flow cytometry. As a result, the concentration of the donor was less than 85% (# 6: 66%, # 7: 71%, # 8: 57%). The method and reagents for this measurement were the same as in comparative example 1.
(step 4-3: analysis of Gene expression in mesenchymal Stem cell derived from bone marrow)
The bone marrow-derived mesenchymal stem cells cultured by the above-described culture method were analyzed for expression of genes (KCNAB1 gene, SULT1E1 gene, MN1 gene, RARRES2 gene) by quantitative PCR. As a result, #6 was the KCNAB1 gene: 0.0015, SULT1E1 gene: undetectable due to too low expression level, MN1 gene: 0.54, RARRES2 gene: 0.0026. #7 is the KCNAB1 gene: 0.0020, SULT1E1 gene: undetectable due to too low expression level, MN1 gene: 0.90, RARRES2 gene: 0.0029. #8 is the KCNAB1 gene: 0.0033, SULT1E1 gene: undetectable due to too low expression level, MN1 gene: 0.52, RARRES2 gene: 0.0033. the method and reagents for this measurement were the same as in example 3.
The results of the quantitative PCR gene expression analysis in example 3 and the reference example are summarized in table 3.
[ Table 3]
Results of quantitative PCR gene expression analysis and flow cytometry analysis described in example 3 and reference example
Figure BDA0002557821570000461
Sequence listing
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Pro Gly Leu Tyr Ala Cys Gly Glu Ala Ala Cys Ala Ser Val His Gly
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Pro Gly Leu Tyr Ala Cys Gly Glu Ala Ala Cys Ala Ser Val His Gly
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Ala Asn Arg Leu Gly Ala Asn Ser Leu Leu Asp Leu Val Val Phe Gly
405 410 415
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Val Pro Pro Ile Lys Pro Asn Ala Gly Glu Glu Ser Val Met Asn Leu
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His Thr Leu Ser Tyr Val Asp Val Gly Thr Gly Lys Val Thr Leu Glu
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Gly Arg Lys Gln Gln Asn Lys Leu Lys Asp Leu Ser Pro Ile Ala Glu
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Arg Leu Gly Cys Thr Leu Pro Gln Leu Ala Val Ala Trp Cys Leu Arg
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His Val Val Asn Glu Ile Asp Asn Ile Leu Arg Asn Lys Pro Tyr Ser
370 375 380
aag aag gac tat aga tca taa 1173
Lys Lys Asp Tyr Arg Ser
385 390
<210>4
<211>390
<212>PRT
<213> Intelligent people
<400>4
Met Leu Ala Ala Arg Thr Gly Ala Ala Gly Ser Gln Ile Ser Glu Glu
1 5 10 15
Asn Thr Lys Leu Arg Arg Gln Ser Gly Phe Ser Val Ala Gly Lys Asp
20 25 30
Lys Ser Pro Lys Lys Ala Ser Glu Asn Ala Lys Asp Ser Ser Leu Ser
35 40 45
Pro Ser Gly Glu Ser Gln Leu Arg Ala Arg Gln Leu Ala Leu Leu Arg
50 55 60
Glu Val Glu Met Asn Trp Tyr Leu Lys Leu Cys Asp Leu Ser Ser Glu
65 70 75 80
His Thr Thr Val Cys Thr Thr Gly Met Pro His Arg Asn Leu Gly Lys
85 90 95
Ser Gly Leu Arg Val Ser Cys Leu Gly Leu Gly Thr Trp Val Thr Phe
100 105 110
Gly Gly Gln Ile Ser Asp Glu Val Ala Glu Arg Leu Met Thr Ile Ala
115 120 125
Tyr Glu Ser Gly Val Asn Leu Phe Asp Thr Ala Glu Val Tyr Ala Ala
130 135 140
Gly Lys Ala Glu Val Ile Leu Gly Ser Ile Ile Lys Lys Lys Gly Trp
145 150 155 160
Arg Arg Ser Ser Leu Val Ile Thr Thr Lys Leu Tyr Trp Gly Gly Lys
165 170 175
Ala Glu Thr Glu Arg Gly Leu Ser Arg Lys His Ile Ile Glu Glu Ile
180 185 190
Val Arg Ala Met Thr His Val Ile Asn Gln Gly Met Ala Met Tyr Trp
195 200 205
Gly Thr Ser Arg Trp Ser Ala Met Glu Ile Met Glu Ala Tyr Ser Val
210 215 220
Ala Arg Gln Phe Asn Met Ile Pro Pro Val Cys Glu Gln Ala Glu Tyr
225 230 235 240
His Leu Phe Gln Arg Glu Lys Val Glu Val Gln Leu Pro Glu Leu Tyr
245 250 255
His Lys Ile Gly Val Gly Ala Met Thr Trp Ser Pro Leu Ala Cys Gly
260 265 270
Ile Ile Ser Gly Lys Tyr Gly Asn Gly Val Pro Glu Ser Ser Arg Ala
275 280 285
Ser Leu Lys Cys Tyr Gln Trp Leu Lys Glu Arg Ile Val Ser Glu Glu
290 295 300
Gly Arg Lys Gln Gln Asn Lys Leu Lys Asp Leu Ser Pro Ile Ala Glu
305 310 315 320
Arg Leu Gly Cys Thr Leu Pro Gln Leu Ala Val Ala Trp Cys Leu Arg
325 330 335
Asn Glu Gly Val Ser Ser Val Leu Leu Gly Ser Ser Thr Pro Glu Gln
340 345 350
Leu Ile Glu Asn Leu Gly Ala Ile Gln Val Leu Pro Lys Met Thr Ser
355 360 365
His Val Val Asn Glu Ile Asp Asn Ile Leu Arg Asn Lys Pro Tyr Ser
370 375 380
Lys Lys Asp Tyr Arg Ser
385 390
<210>5
<211>885
<212>DNA
<213> Intelligent people
<220>
<221>CDS
<222>(1)..(885)
<400>5
atg aat tct gaa ctt gac tat tat gaa aag ttt gaa gaa gtc cat ggg 48
Met Asn Ser Glu Leu Asp Tyr Tyr Glu Lys Phe Glu Glu Val His Gly
1 5 10 15
att cta atg tat aaa gat ttt gtc aaa tat tgg gat aat gtg gaa gcg 96
Ile Leu Met Tyr Lys Asp Phe Val Lys Tyr Trp Asp Asn Val Glu Ala
20 25 30
ttc cag gca aga cca gat gat ctt gtc att gcc acc tac cct aaa tct 144
Phe Gln Ala Arg Pro Asp Asp Leu Val Ile Ala Thr Tyr Pro Lys Ser
35 40 45
ggt aca acc tgg gtt agt gaa att gtg tat atg atc tat aaa gag ggt 192
Gly Thr Thr Trp Val Ser Glu Ile Val Tyr Met Ile Tyr Lys Glu Gly
50 55 60
gat gtg gaa aag tgc aaa gaa gat gta att ttt aat cga ata cct ttc 240
Asp Val Glu Lys Cys Lys Glu Asp Val Ile Phe Asn Arg Ile Pro Phe
65 70 75 80
ctg gaa tgc aga aaa gaa aac ctc atg aat gga gta aaa caa tta gat 288
Leu Glu Cys Arg Lys Glu Asn Leu Met Asn Gly Val Lys Gln Leu Asp
85 90 95
gag atg aat tct cct aga att gtg aag act cat ttg cca cct gaa ctt 336
Glu Met Asn Ser Pro Arg Ile Val Lys Thr His Leu Pro Pro Glu Leu
100 105 110
ctt cct gcc tca ttt tgg gaa aag gat tgt aag ata atc tat ctt tgc 384
Leu Pro Ala Ser Phe Trp Glu Lys Asp Cys Lys Ile Ile Tyr Leu Cys
115 120 125
cgg aat gca aag gat gtg gct gtt tcc ttt tat tat ttc ttt cta atg 432
Arg Asn Ala Lys Asp Val Ala Val Ser Phe Tyr Tyr Phe Phe Leu Met
130 135 140
gtg gct ggt cat cca aat cct gga tcc ttt cca gag ttt gtg gag aaa 480
Val Ala Gly His Pro Asn Pro Gly Ser Phe Pro Glu Phe Val Glu Lys
145 150 155 160
ttc atg caa gga cag gtt cct tat ggt tcc tgg tat aaa cat gta aaa 528
Phe Met Gln Gly Gln Val Pro Tyr Gly Ser Trp Tyr Lys His Val Lys
165 170 175
tct tgg tgg gaa aag gga aag agt cca cgt gta cta ttt ctt ttc tac 576
Ser Trp Trp Glu Lys Gly Lys Ser Pro Arg Val Leu Phe Leu Phe Tyr
180 185 190
gaa gac ctg aaa gag gat atc aga aaa gag gtg ata aaa ttg ata cat 624
Glu Asp Leu Lys Glu Asp Ile Arg Lys Glu Val Ile Lys Leu Ile His
195 200 205
ttc ctg gaa agg aag cca tca gag gag ctt gtg gac agg att ata cat 672
Phe Leu Glu Arg Lys Pro Ser Glu Glu Leu Val Asp Arg Ile Ile His
210 215 220
cat act tcg ttc caa gag atg aag aac aat cca tcc aca aat tac aca 720
His Thr Ser Phe Gln Glu Met Lys Asn Asn Pro Ser Thr Asn Tyr Thr
225 230 235 240
aca ctg cca gac gaa att atg aac cag aaa ttg tcg ccc ttc atg aga 768
Thr Leu Pro Asp Glu Ile Met Asn Gln Lys Leu Ser Pro Phe Met Arg
245 250 255
aag gga att aca gga gac tgg aaa aat cac ttt aca gta gcc ctg aat 816
Lys Gly Ile Thr Gly Asp Trp Lys Asn His Phe Thr Val Ala Leu Asn
260 265 270
gaa aaa ttt gat aaa cat tat gag cag caa atg aag gaa tct aca ctg 864
Glu Lys Phe Asp Lys His Tyr Glu Gln Gln Met Lys Glu Ser Thr Leu
275 280 285
aag ttt cga act gag atc taa 885
Lys Phe Arg Thr Glu Ile
290
<210>6
<211>294
<212>PRT
<213> Intelligent people
<400>6
Met Asn Ser Glu Leu Asp Tyr Tyr Glu Lys Phe Glu Glu Val His Gly
1 5 10 15
Ile Leu Met Tyr Lys Asp Phe Val Lys Tyr Trp Asp Asn Val Glu Ala
2025 30
Phe Gln Ala Arg Pro Asp Asp Leu Val Ile Ala Thr Tyr Pro Lys Ser
35 40 45
Gly Thr Thr Trp Val Ser Glu Ile Val Tyr Met Ile Tyr Lys Glu Gly
50 55 60
Asp Val Glu Lys Cys Lys Glu Asp Val Ile Phe Asn Arg Ile Pro Phe
65 70 75 80
Leu Glu Cys Arg Lys Glu Asn Leu Met Asn Gly Val Lys Gln Leu Asp
85 90 95
Glu Met Asn Ser Pro Arg Ile Val Lys Thr His Leu Pro Pro Glu Leu
100 105 110
Leu Pro Ala Ser Phe Trp Glu Lys Asp Cys Lys Ile Ile Tyr Leu Cys
115 120 125
Arg Asn Ala Lys Asp Val Ala Val Ser Phe Tyr Tyr Phe Phe Leu Met
130 135 140
Val Ala Gly His Pro Asn Pro Gly Ser Phe Pro Glu Phe Val Glu Lys
145 150 155 160
Phe Met Gln Gly Gln Val Pro Tyr Gly Ser Trp Tyr Lys His Val Lys
165 170 175
Ser Trp Trp Glu Lys Gly Lys Ser Pro Arg Val Leu Phe Leu Phe Tyr
180185 190
Glu Asp Leu Lys Glu Asp Ile Arg Lys Glu Val Ile Lys Leu Ile His
195 200 205
Phe Leu Glu Arg Lys Pro Ser Glu Glu Leu Val Asp Arg Ile Ile His
210 215 220
His Thr Ser Phe Gln Glu Met Lys Asn Asn Pro Ser Thr Asn Tyr Thr
225 230 235 240
Thr Leu Pro Asp Glu Ile Met Asn Gln Lys Leu Ser Pro Phe Met Arg
245 250 255
Lys Gly Ile Thr Gly Asp Trp Lys Asn His Phe Thr Val Ala Leu Asn
260 265 270
Glu Lys Phe Asp Lys His Tyr Glu Gln Gln Met Lys Glu Ser Thr Leu
275 280 285
Lys Phe Arg Thr Glu Ile
290
<210>7
<211>3963
<212>DNA
<213> Intelligent people
<220>
<221>CDS
<222>(1)..(3963)
<400>7
atg ttt ggg ctg gac caa ttc gag ccc cag gtc aac agc agg aac gct 48
Met Phe Gly Leu Asp Gln Phe Glu Pro Gln Val Asn Ser Arg Asn Ala
1 5 10 15
ggc cag ggc gag agg aac ttt aac gag acc gga ctg agc atg aac acc 96
Gly Gln Gly Glu Arg Asn Phe Asn Glu Thr Gly Leu Ser Met Asn Thr
20 25 30
cac ttt aag gcc ccg gct ttc cac act ggg ggg ccc cct ggc cct gtg 144
His Phe Lys Ala Pro Ala Phe His Thr Gly Gly Pro Pro Gly Pro Val
35 40 45
gat cct gct atg agc gcg ctg ggc gaa ccc ccg atc ttg ggc atg aac 192
Asp Pro Ala Met Ser Ala Leu Gly Glu Pro Pro Ile Leu Gly Met Asn
50 55 60
atg gag ccc tac ggc ttc cac gcg cgc ggc cac tcg gag ttg cac gca 240
Met Glu Pro Tyr Gly Phe His Ala Arg Gly His Ser Glu Leu His Ala
65 70 75 80
ggg ggg ctg caa gcg cag cct gtg cac ggc ttc ttt ggc ggc cag cag 288
Gly Gly Leu Gln Ala Gln Pro Val His Gly Phe Phe Gly Gly Gln Gln
85 90 95
cct cac cac ggc cac ccg gga agt cat cat ccc cac cag cat cac ccc 336
Pro His His Gly His Pro Gly Ser His His Pro His Gln His His Pro
100 105 110
cac ttt ggg ggc aac ttc ggt ggcccg gac ccc ggg gcc tcg tgc ctg 384
His Phe Gly Gly Asn Phe Gly Gly Pro Asp Pro Gly Ala Ser Cys Leu
115 120 125
cac ggg ggt cgc ctg ctc ggc tac ggc ggc gca gcc gga ggc ctg ggc 432
His Gly Gly Arg Leu Leu Gly Tyr Gly Gly Ala Ala Gly Gly Leu Gly
130 135 140
agc cag ccg ccc ttc gcc gag ggc tat gag cac atg gcg gag agc cag 480
Ser Gln Pro Pro Phe Ala Glu Gly Tyr Glu His Met Ala Glu Ser Gln
145 150 155 160
ggg cct gag agc ttc ggc ccg cag cga ccg ggg aac ctc ccg gac ttc 528
Gly Pro Glu Ser Phe Gly Pro Gln Arg Pro Gly Asn Leu Pro Asp Phe
165 170 175
cac agt tca ggt gcc tcc agc cac gcc gtg ccg gcc cca tgc ctg ccg 576
His Ser Ser Gly Ala Ser Ser His Ala Val Pro Ala Pro Cys Leu Pro
180 185 190
ctg gac cag agc cct aac cga gcc gcc tcc ttc cac ggc ctg ccg tcc 624
Leu Asp Gln Ser Pro Asn Arg Ala Ala Ser Phe His Gly Leu Pro Ser
195 200 205
tcc agc ggc tcc gat tcc cac agt ctg gag cca cgg agg gtg acg aac 672
Ser Ser Gly Ser Asp Ser His Ser Leu Glu Pro Arg Arg Val Thr Asn
210 215 220
caa gga gcc gtc gac tcg ctg gaa tac aat tac ccg ggc gag gcg ccc 720
Gln Gly Ala Val Asp Ser Leu Glu Tyr Asn Tyr Pro Gly Glu Ala Pro
225 230 235 240
tcg gga cat ttt gac atg ttt tcg ccc tct gac tcc gaa ggg cag ctg 768
Ser Gly His Phe Asp Met Phe Ser Pro Ser Asp Ser Glu Gly Gln Leu
245 250 255
cct cat tat gca gcg ggt cgc cag gtt cct ggg ggc gct ttc ccg ggc 816
Pro His Tyr Ala Ala Gly Arg Gln Val Pro Gly Gly Ala Phe Pro Gly
260 265 270
gcc tcg gcc atg ccc aga gct gcg ggc atg gtg ggc ttg tcc aaa atg 864
Ala Ser Ala Met Pro Arg Ala Ala Gly Met Val Gly Leu Ser Lys Met
275 280 285
cac gcc cag cca ccg cag cag cag ccc cag cag cag cag cag ccc cag 912
His Ala Gln Pro Pro Gln Gln Gln Pro Gln Gln Gln Gln Gln Pro Gln
290 295 300
cag cag cag cag cag cat ggt gtg ttc ttt gag agg ttc agt ggg gcc 960
Gln Gln Gln Gln Gln His Gly Val Phe Phe Glu Arg Phe Ser Gly Ala
305 310 315 320
aga aag atg cct gtg ggt ctg gag ccc tca gtg ggctcc agg cac ccg 1008
Arg Lys Met Pro Val Gly Leu Glu Pro Ser Val Gly Ser Arg His Pro
325 330 335
tta atg cag cct ccc cag cag gcc ccg cca ccc cct cag cag cag ccc 1056
Leu Met Gln Pro Pro Gln Gln Ala Pro Pro Pro Pro Gln Gln Gln Pro
340 345 350
ccg cag cag ccg cca cag cag cag ccg ccg ccg cca ccc ggg ctt cta 1104
Pro Gln Gln Pro Pro Gln Gln Gln Pro Pro Pro Pro Pro Gly Leu Leu
355 360 365
gtc cga caa aat tcg tgc ccg cct gcg ctc cct cgg ccc cag cag ggc 1152
Val Arg Gln Asn Ser Cys Pro Pro Ala Leu Pro Arg Pro Gln Gln Gly
370 375 380
gag gcg ggc acg ccc agc ggc ggc ctg cag gac gga ggc ccc atg ctg 1200
Glu Ala Gly Thr Pro Ser Gly Gly Leu Gln Asp Gly Gly Pro Met Leu
385 390 395 400
ccc agc cag cac gcg caa ttc gag tat ccc atc cac cgg ctg gag aac 1248
Pro Ser Gln His Ala Gln Phe Glu Tyr Pro Ile His Arg Leu Glu Asn
405 410 415
cgg agc atg cac cct tat tcc gag cct gtt ttc agc atg cag cat cct 1296
Arg Ser Met His Pro Tyr Ser Glu Pro Val Phe Ser Met Gln His Pro
420 425 430
cct ccg cag cag gcg ccc aac cag cgg ctg cag cat ttc gac gcg ccc 1344
Pro Pro Gln Gln Ala Pro Asn Gln Arg Leu Gln His Phe Asp Ala Pro
435 440 445
ccc tac atg aac gtg gcc aag agg ccg cgc ttc gac ttt ccg ggc agc 1392
Pro Tyr Met Asn Val Ala Lys Arg Pro Arg Phe Asp Phe Pro Gly Ser
450 455 460
gcg gga gtg gac cgc tgc gct tcg tgg aac ggc agc atg cac aac ggc 1440
Ala Gly Val Asp Arg Cys Ala Ser Trp Asn Gly Ser Met His Asn Gly
465 470 475 480
gct ctg gat aat cac ctc tcc cct tcc gcc tac cca ggc cta ccc ggc 1488
Ala Leu Asp Asn His Leu Ser Pro Ser Ala Tyr Pro Gly Leu Pro Gly
485 490 495
gag ttc aca ccg cct gtg ccc gac agc ttc cct tcg ggg ccg ccc ctg 1536
Glu Phe Thr Pro Pro Val Pro Asp Ser Phe Pro Ser Gly Pro Pro Leu
500 505 510
cag cat ccg gcc ccg gac cac cag tcc ctg caa cag cag cag cag cag 1584
Gln His Pro Ala Pro Asp His Gln Ser Leu Gln Gln Gln Gln Gln Gln
515 520 525
cag cag cag cag caa cag cag cag cag cag cag caa cag caa cag caa 1632
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
530 535 540
cag cag cag cag cag cag cgc caa aac gcg gcc ctc atg att aag cag 1680
Gln Gln Gln Gln Gln Gln Arg Gln Asn Ala Ala Leu Met Ile Lys Gln
545 550 555 560
atg gcg tcg cgg aat cag cag cag cgg ctg cgc cag ccc aac ctg gct 1728
Met Ala Ser Arg Asn Gln Gln Gln Arg Leu Arg Gln Pro Asn Leu Ala
565 570 575
cag cta ggc cac ccc ggg gac gtg ggc cag ggc ggc ctg gtg cat ggc 1776
Gln Leu Gly His Pro Gly Asp Val Gly Gln Gly Gly Leu Val His Gly
580 585 590
ggc ccg gtg ggc ggc ttg gcc cag ccg aac ttt gag cgc gaa ggc ggc 1824
Gly Pro Val Gly Gly Leu Ala Gln Pro Asn Phe Glu Arg Glu Gly Gly
595 600 605
agc acg ggc gcc ggg cgt ctg ggc acc ttc gag cag cag gcg ccg cac 1872
Ser Thr Gly Ala Gly Arg Leu Gly Thr Phe Glu Gln Gln Ala Pro His
610 615 620
ttg gcg caa gag agc gcg tgg ttc tca ggt ccg cat ccg ccg ccc gga 1920
Leu Ala Gln Glu Ser Ala Trp Phe Ser Gly Pro His Pro Pro Pro Gly
625 630 635 640
gac ctg ctg ccc cgt agg atg ggc ggc tcg ggt ctg ccc gct gac tgt 1968
Asp Leu Leu Pro Arg Arg Met Gly Gly Ser Gly Leu Pro Ala Asp Cys
645 650 655
ggc ccg cac gac ccc agc ctg gcg ccc cct cct ccg cct ggt ggc tcg 2016
Gly Pro His Asp Pro Ser Leu Ala Pro Pro Pro Pro Pro Gly Gly Ser
660 665 670
ggg gtg ctg ttc cgg ggc cct ctg cag gag ccg atg agg atg ccc gga 2064
Gly Val Leu Phe Arg Gly Pro Leu Gln Glu Pro Met Arg Met Pro Gly
675 680 685
gag ggc cac gtg ccc gcg ctg cct tca ccg ggc ctg cag ttc ggg ggc 2112
Glu Gly His Val Pro Ala Leu Pro Ser Pro Gly Leu Gln Phe Gly Gly
690 695 700
agt ctg gga ggc ctg ggt cag ctg cag tcg ccc ggg gcg ggc gtg ggg 2160
Ser Leu Gly Gly Leu Gly Gln Leu Gln Ser Pro Gly Ala Gly Val Gly
705 710 715 720
ctc ccc agc gct gct tcg gag cgc cgg ccc ccg ccg ccg gac ttt gct 2208
Leu Pro Ser Ala Ala Ser Glu Arg Arg Pro Pro Pro Pro Asp Phe Ala
725 730 735
acg tct gcg ctc ggg ggc cag ccg ggc ttt ccg ttt ggt gca gcc ggc 2256
Thr Ser Ala Leu Gly Gly Gln Pro Gly Phe Pro Phe Gly Ala Ala Gly
740 745 750
cgg cag tcc acg ccg cac agc ggt cca ggc gtg aac tcg ccc ccc agc 2304
Arg Gln Ser Thr Pro His Ser Gly Pro Gly Val Asn Ser Pro Pro Ser
755 760 765
gcg gga ggg ggc ggt ggc agc tct ggt ggc ggc ggt ggc ggg ggt gcc 2352
Ala Gly Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Gly Gly Gly Ala
770 775 780
tac ccg ccg cag cct gat ttc cag ccc agc cag cgc acc tcg gcc agt 2400
Tyr Pro Pro Gln Pro Asp Phe Gln Pro Ser Gln Arg Thr Ser Ala Ser
785 790 795 800
aaa ttg ggc gcg ctc tcg ctg ggc tcc ttc aac aag ccc agc tcc aag 2448
Lys Leu Gly Ala Leu Ser Leu Gly Ser Phe Asn Lys Pro Ser Ser Lys
805 810 815
gac aac ctg ttc ggc cag agc tgc ctg gct gcg ctc tcc acc gct tgc 2496
Asp Asn Leu Phe Gly Gln Ser Cys Leu Ala Ala Leu Ser Thr Ala Cys
820 825 830
cag aac atg atc gcc agc ctc ggg gcc ccc aac ctc aac gtg acc ttc 2544
Gln Asn Met Ile Ala Ser Leu Gly Ala Pro Asn Leu Asn Val Thr Phe
835 840 845
aac aag aag aac ccg cca gag ggc aag agg aaa ctg agc cag aac gag 2592
Asn Lys Lys Asn Pro Pro Glu Gly Lys Arg Lys Leu Ser Gln Asn Glu
850 855 860
acc gac ggc gcg gca gtg gcc ggc aac ccg ggc tcg gat tac ttc cca 2640
Thr Asp Gly Ala Ala Val Ala Gly Asn Pro Gly Ser Asp Tyr Phe Pro
865 870 875 880
gga ggg act gct cct ggg gcc cca gga ccc gga ggc ccg tcc ggg acc 2688
Gly Gly Thr Ala Pro Gly Ala Pro Gly Pro Gly Gly Pro Ser Gly Thr
885 890 895
agt agc agc ggc tcc aaa gcc tcg ggg ccg ccc aac cct cca gcc cag 2736
Ser Ser Ser Gly Ser Lys Ala Ser Gly Pro Pro Asn Pro Pro Ala Gln
900 905 910
ggg gac ggc acc agc ctc tcc ccc aac tac acc ctg gaa tcc acg tcg 2784
Gly Asp Gly Thr Ser Leu Ser Pro Asn Tyr Thr Leu Glu Ser Thr Ser
915 920 925
ggg aat gac ggc aag ccg gtc tcc ggg ggc ggc ggc cgg gga cgg ggt 2832
Gly Asn Asp Gly Lys Pro Val Ser Gly Gly Gly Gly Arg Gly Arg Gly
930 935 940
cgc aga aaa agg gac agt ggt cac gtg agc cct ggc acc ttc tttgac 2880
Arg Arg Lys Arg Asp Ser Gly His Val Ser Pro Gly Thr Phe Phe Asp
945 950 955 960
aag tac tcg gcg gct ccg gac agc ggg ggc gca cct ggg gtg agc cca 2928
Lys Tyr Ser Ala Ala Pro Asp Ser Gly Gly Ala Pro Gly Val Ser Pro
965 970 975
ggg cag cag caa gcg tca ggc gca gcc gtc ggg gga agc tcc gca ggc 2976
Gly Gln Gln Gln Ala Ser Gly Ala Ala Val Gly Gly Ser Ser Ala Gly
980 985 990
gag acg cgc ggg gca ccg acg ccc cac gaa aag gcg ctc acg tcg cca 3024
Glu Thr Arg Gly Ala Pro Thr Pro His Glu Lys Ala Leu Thr Ser Pro
995 1000 1005
tcc tgg ggg aag ggg gct gag ttg ctc ctg ggg gat cag ccg gac 3069
Ser Trp Gly Lys Gly Ala Glu Leu Leu Leu Gly Asp Gln Pro Asp
1010 1015 1020
ctc att ggg tcc ctg gac ggc ggg gcc aag tcg gac agt agt tcg 3114
Leu Ile Gly Ser Leu Asp Gly Gly Ala Lys Ser Asp Ser Ser Ser
1025 1030 1035
cca aac gtg ggt gag ttc gcc tcg gac gag gtg agc acg agc tac 3159
Pro Asn Val Gly Glu Phe Ala Ser Asp Glu Val Ser Thr Ser Tyr
1040 10451050
gcc aat gag gac gag gtg tcg tcc agc tct gac aac ccc cag gca 3204
Ala Asn Glu Asp Glu Val Ser Ser Ser Ser Asp Asn Pro Gln Ala
1055 1060 1065
cta gtt aaa gcg agc agg agt ccc ctg gtg acc ggc tcg ccc aaa 3249
Leu Val Lys Ala Ser Arg Ser Pro Leu Val Thr Gly Ser Pro Lys
1070 1075 1080
ctc cct ccc cgt ggg gta ggc gcc ggg gaa cac gga ccg aag gcg 3294
Leu Pro Pro Arg Gly Val Gly Ala Gly Glu His Gly Pro Lys Ala
1085 1090 1095
ccc ccg ccc gcc ctc ggc ctg ggc atc atg tct aac tct acc tcg 3339
Pro Pro Pro Ala Leu Gly Leu Gly Ile Met Ser Asn Ser Thr Ser
1100 1105 1110
acc cct gac agc tac ggc ggc ggt ggg ggc ccg ggc cat ccg ggc 3384
Thr Pro Asp Ser Tyr Gly Gly Gly Gly Gly Pro Gly His Pro Gly
1115 1120 1125
act ccg ggc ctg gag cag gtc cgc acc ccg acg agc agc agc ggc 3429
Thr Pro Gly Leu Glu Gln Val Arg Thr Pro Thr Ser Ser Ser Gly
1130 1135 1140
gcc ccg cca ccc gac gag atc cac ccc ctg gag atc ctt cag gcg 3474
Ala Pro Pro Pro Asp Glu Ile His Pro Leu Glu Ile Leu Gln Ala
1145 1150 1155
cag atc cag cta cag agg cag cag ttc agc atc tcc gag gac cag 3519
Gln Ile Gln Leu Gln Arg Gln Gln Phe Ser Ile Ser Glu Asp Gln
1160 1165 1170
cct ctg ggg ctg aag ggt ggc aag aag ggt gag tgc gcc gtc ggg 3564
Pro Leu Gly Leu Lys Gly Gly Lys Lys Gly Glu Cys Ala Val Gly
1175 1180 1185
gcc tca ggg gcg cag aat ggc gac agc gag ctg ggc agc tgc tgc 3609
Ala Ser Gly Ala Gln Asn Gly Asp Ser Glu Leu Gly Ser Cys Cys
1190 1195 1200
tcc gag gcg gtc aag agc gcc atg agc acc att gac ctg gac tcg 3654
Ser Glu Ala Val Lys Ser Ala Met Ser Thr Ile Asp Leu Asp Ser
1205 1210 1215
ctg atg gca gag cac agc gct gcc tgg tac atg ccc gct gac aag 3699
Leu Met Ala Glu His Ser Ala Ala Trp Tyr Met Pro Ala Asp Lys
1220 1225 1230
gcc ctg gtg gac agc gcg gac gac gac aag acg ttg gcg ccc tgg 3744
Ala Leu Val Asp Ser Ala Asp Asp Asp Lys Thr Leu Ala Pro Trp
1235 1240 1245
gag aag gcc aaa ccc cag aac ccc aac agc aaa gaa gcc cac gac 3789
Glu Lys Ala Lys Pro Gln Asn Pro Asn Ser Lys Glu Ala His Asp
1250 1255 1260
ctc cct gca aac aag gcc tca gca tcc cag cct ggc agc cac ttg 3834
Leu Pro Ala Asn Lys Ala Ser Ala Ser Gln Pro Gly Ser His Leu
1265 1270 1275
cag tgc ctg tct gtc cac tgc aca gac gac gtg ggt gac gcc aag 3879
Gln Cys Leu Ser Val His Cys Thr Asp Asp Val Gly Asp Ala Lys
1280 1285 1290
gct cga gcc tcc gtg ccc acc tgg cgg tcc ctg cat tct gac atc 3924
Ala Arg Ala Ser Val Pro Thr Trp Arg Ser Leu His Ser Asp Ile
1295 1300 1305
tcc aac aga ttt ggg aca ttc gtg gct gcc cta act tga 3963
Ser Asn Arg Phe Gly Thr Phe Val Ala Ala Leu Thr
1310 1315 1320
<210>8
<211>1320
<212>PRT
<213> Intelligent people
<400>8
Met Phe Gly Leu Asp Gln Phe Glu Pro Gln Val Asn Ser Arg Asn Ala
1 5 10 15
Gly Gln Gly Glu Arg Asn Phe Asn Glu Thr Gly Leu Ser Met Asn Thr
20 25 30
His Phe Lys Ala Pro Ala Phe His Thr Gly Gly Pro Pro Gly Pro Val
35 40 45
Asp Pro Ala Met Ser Ala Leu Gly Glu Pro Pro Ile Leu Gly Met Asn
50 55 60
Met Glu Pro Tyr Gly Phe His Ala Arg Gly His Ser Glu Leu His Ala
65 70 75 80
Gly Gly Leu Gln Ala Gln Pro Val His Gly Phe Phe Gly Gly Gln Gln
85 90 95
Pro His His Gly His Pro Gly Ser His His Pro His Gln His His Pro
100 105 110
His Phe Gly Gly Asn Phe Gly Gly Pro Asp Pro Gly Ala Ser Cys Leu
115 120 125
His Gly Gly Arg Leu Leu Gly Tyr Gly Gly Ala Ala Gly Gly Leu Gly
130 135 140
Ser Gln Pro Pro Phe Ala Glu Gly Tyr Glu His Met Ala Glu Ser Gln
145 150 155 160
Gly Pro Glu Ser Phe Gly Pro Gln Arg Pro Gly Asn Leu Pro Asp Phe
165 170 175
His Ser Ser Gly Ala Ser Ser His Ala Val Pro Ala Pro Cys Leu Pro
180 185 190
Leu Asp Gln Ser Pro Asn Arg Ala Ala Ser Phe His Gly Leu Pro Ser
195 200 205
Ser Ser Gly Ser Asp Ser His Ser Leu Glu Pro Arg Arg Val Thr Asn
210 215 220
Gln Gly Ala Val Asp Ser Leu Glu Tyr Asn Tyr Pro Gly Glu Ala Pro
225 230 235 240
Ser Gly His Phe Asp Met Phe Ser Pro Ser Asp Ser Glu Gly Gln Leu
245 250 255
Pro His Tyr Ala Ala Gly Arg Gln Val Pro Gly Gly Ala Phe Pro Gly
260 265 270
Ala Ser Ala Met Pro Arg Ala Ala Gly Met Val Gly Leu Ser Lys Met
275 280 285
His Ala Gln Pro Pro Gln Gln Gln Pro Gln Gln Gln Gln Gln Pro Gln
290 295 300
Gln Gln Gln Gln Gln His Gly Val Phe Phe Glu Arg Phe Ser Gly Ala
305 310 315 320
Arg Lys Met Pro Val Gly Leu Glu Pro Ser Val Gly Ser Arg His Pro
325 330 335
Leu Met Gln Pro Pro Gln Gln Ala Pro Pro Pro Pro Gln Gln Gln Pro
340 345 350
Pro Gln Gln Pro Pro Gln Gln Gln Pro Pro Pro Pro Pro Gly Leu Leu
355 360 365
Val Arg Gln Asn Ser Cys Pro Pro Ala Leu Pro Arg Pro Gln Gln Gly
370 375 380
Glu Ala Gly Thr Pro Ser Gly Gly Leu Gln Asp Gly Gly Pro Met Leu
385 390 395 400
Pro Ser Gln His Ala Gln Phe Glu Tyr Pro Ile His Arg Leu Glu Asn
405 410 415
Arg Ser Met His Pro Tyr Ser Glu Pro Val Phe Ser Met Gln His Pro
420 425 430
Pro Pro Gln Gln Ala Pro Asn Gln Arg Leu Gln His Phe Asp Ala Pro
435 440 445
Pro Tyr Met Asn Val Ala Lys Arg Pro Arg Phe Asp Phe Pro Gly Ser
450 455 460
Ala Gly Val Asp Arg Cys Ala Ser Trp Asn Gly Ser Met His Asn Gly
465 470 475 480
Ala Leu Asp Asn His Leu Ser Pro Ser Ala Tyr Pro Gly Leu Pro Gly
485 490 495
Glu Phe Thr Pro Pro Val Pro Asp Ser Phe Pro Ser Gly Pro Pro Leu
500 505 510
Gln His Pro Ala Pro Asp His Gln Ser Leu Gln Gln Gln Gln Gln Gln
515 520 525
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
530 535 540
Gln Gln Gln Gln Gln Gln Arg Gln Asn Ala Ala Leu Met Ile Lys Gln
545 550 555 560
Met Ala Ser Arg Asn Gln Gln Gln Arg Leu Arg Gln Pro Asn Leu Ala
565 570 575
Gln Leu Gly His Pro Gly Asp Val Gly Gln Gly Gly Leu Val His Gly
580 585 590
Gly Pro Val Gly Gly Leu Ala Gln Pro Asn Phe Glu Arg Glu Gly Gly
595 600 605
Ser Thr Gly Ala Gly Arg Leu Gly Thr Phe Glu Gln Gln Ala Pro His
610 615 620
Leu Ala Gln Glu Ser Ala Trp Phe Ser Gly Pro His Pro Pro Pro Gly
625 630 635 640
Asp Leu Leu Pro Arg Arg Met Gly Gly Ser Gly Leu Pro Ala Asp Cys
645 650 655
Gly Pro His Asp Pro Ser Leu Ala Pro Pro Pro Pro Pro Gly Gly Ser
660 665 670
Gly Val Leu Phe Arg Gly Pro Leu Gln Glu Pro Met Arg Met Pro Gly
675 680 685
Glu Gly His Val Pro Ala Leu Pro Ser Pro Gly Leu Gln Phe Gly Gly
690 695 700
Ser Leu Gly Gly Leu Gly Gln Leu Gln Ser Pro Gly Ala Gly Val Gly
705 710 715 720
Leu Pro Ser Ala Ala Ser Glu Arg Arg Pro Pro Pro Pro Asp Phe Ala
725 730 735
Thr Ser Ala Leu Gly Gly Gln Pro Gly Phe Pro Phe Gly Ala Ala Gly
740 745 750
Arg Gln Ser Thr Pro His Ser Gly Pro Gly Val Asn Ser Pro Pro Ser
755 760 765
Ala Gly Gly Gly Gly Gly Ser Ser Gly Gly Gly Gly Gly Gly Gly Ala
770 775 780
Tyr Pro Pro Gln Pro Asp Phe Gln Pro Ser Gln Arg Thr Ser Ala Ser
785 790 795 800
Lys Leu Gly Ala Leu Ser Leu Gly Ser Phe Asn Lys Pro Ser Ser Lys
805 810 815
Asp Asn Leu Phe Gly Gln Ser Cys Leu Ala Ala Leu Ser Thr Ala Cys
820 825 830
Gln Asn Met Ile Ala Ser Leu Gly Ala Pro Asn Leu Asn Val Thr Phe
835 840 845
Asn Lys Lys Asn Pro Pro Glu Gly Lys Arg Lys Leu Ser Gln Asn Glu
850 855 860
Thr Asp Gly Ala Ala Val Ala Gly Asn Pro Gly Ser Asp Tyr Phe Pro
865 870 875 880
Gly Gly Thr Ala Pro Gly Ala Pro Gly Pro Gly Gly Pro Ser Gly Thr
885 890 895
Ser Ser Ser Gly Ser Lys Ala Ser Gly Pro Pro Asn Pro Pro Ala Gln
900 905 910
Gly Asp Gly Thr Ser Leu Ser Pro Asn Tyr Thr Leu Glu Ser Thr Ser
915 920 925
Gly Asn Asp Gly Lys Pro Val Ser Gly Gly Gly Gly Arg Gly Arg Gly
930 935 940
Arg Arg Lys Arg Asp Ser Gly His Val Ser Pro Gly Thr Phe Phe Asp
945 950 955 960
Lys Tyr Ser Ala Ala Pro Asp Ser Gly Gly Ala Pro Gly Val Ser Pro
965 970 975
Gly Gln Gln Gln Ala Ser Gly Ala Ala Val Gly Gly Ser Ser Ala Gly
980 985 990
Glu Thr Arg Gly Ala Pro Thr Pro His Glu Lys Ala Leu Thr Ser Pro
995 1000 1005
Ser Trp Gly Lys Gly Ala Glu Leu Leu Leu Gly Asp Gln Pro Asp
1010 1015 1020
Leu Ile Gly Ser Leu Asp Gly Gly Ala Lys Ser Asp Ser Ser Ser
1025 1030 1035
Pro Asn Val Gly Glu Phe Ala Ser Asp Glu Val Ser Thr Ser Tyr
1040 1045 1050
Ala Asn Glu Asp Glu Val Ser Ser Ser Ser Asp Asn Pro Gln Ala
1055 1060 1065
Leu Val Lys Ala Ser Arg Ser Pro Leu Val Thr Gly Ser Pro Lys
1070 1075 1080
Leu Pro Pro Arg Gly Val Gly Ala Gly Glu His Gly Pro Lys Ala
1085 1090 1095
Pro Pro Pro Ala Leu Gly Leu Gly Ile Met Ser Asn Ser Thr Ser
1100 1105 1110
Thr Pro Asp Ser Tyr Gly Gly Gly Gly Gly Pro Gly His Pro Gly
1115 1120 1125
Thr Pro Gly Leu Glu Gln Val Arg Thr Pro Thr Ser Ser Ser Gly
1130 1135 1140
Ala Pro Pro Pro Asp Glu Ile His Pro Leu Glu Ile Leu Gln Ala
1145 1150 1155
Gln Ile Gln Leu Gln Arg Gln Gln Phe Ser Ile Ser Glu Asp Gln
1160 1165 1170
Pro Leu Gly Leu Lys Gly Gly Lys Lys Gly Glu Cys Ala Val Gly
1175 1180 1185
Ala Ser Gly Ala Gln Asn Gly Asp Ser Glu Leu Gly Ser Cys Cys
1190 1195 1200
Ser Glu Ala Val Lys Ser Ala Met Ser Thr Ile Asp Leu Asp Ser
1205 1210 1215
Leu Met Ala Glu His Ser Ala Ala Trp Tyr Met Pro Ala Asp Lys
1220 1225 1230
Ala Leu Val Asp Ser Ala Asp Asp Asp Lys Thr Leu Ala Pro Trp
1235 1240 1245
Glu Lys Ala Lys Pro Gln Asn Pro Asn Ser Lys Glu Ala His Asp
1250 1255 1260
Leu Pro Ala Asn Lys Ala Ser Ala Ser Gln Pro Gly Ser His Leu
1265 1270 1275
Gln Cys Leu Ser Val His Cys Thr Asp Asp Val Gly Asp Ala Lys
1280 1285 1290
Ala Arg Ala Ser Val Pro Thr Trp Arg Ser Leu His Ser Asp Ile
1295 1300 1305
Ser Asn Arg Phe Gly Thr Phe Val Ala Ala Leu Thr
1310 1315 1320
<210>9
<211>399
<212>DNA
<213> Intelligent people
<220>
<221>CDS
<222>(1)..(399)
<400>9
atg cga cgg ctg ctg atc cct ctg gcc ctg tgg ctg ggt gcg gtg ggc 48
Met Arg Arg Leu Leu Ile Pro Leu Ala Leu Trp Leu Gly Ala Val Gly
1 5 10 15
gtg ggc gtc gcc gag ctc acg gaa gcc cag cgc cgg ggc ctg cag gtg 96
Val Gly Val Ala Glu Leu Thr Glu Ala Gln Arg Arg Gly Leu Gln Val
20 25 30
gcc ctg gag gaa ttt cac aag cac ccg ccc gtg cag tgg gcc ttc cag 144
Ala Leu Glu Glu Phe His Lys His Pro Pro Val Gln Trp Ala Phe Gln
35 40 45
gag acc agt gtg gag agc gcc gtg gac acg ccc ttc cca gct gga ata 192
Glu Thr Ser Val Glu Ser Ala Val Asp Thr Pro Phe Pro Ala Gly Ile
50 55 60
ttt gtg agg ctg gaa ttt aag ctg cag cag aca agc tgc cgg aag agg 240
Phe Val Arg Leu Glu Phe Lys Leu Gln Gln Thr Ser Cys Arg Lys Arg
65 7075 80
gac tgg aag aaa ccc gag tgc aaa gtc agg ccc aat ggg agg aaa cgg 288
Asp Trp Lys Lys Pro Glu Cys Lys Val Arg Pro Asn Gly Arg Lys Arg
85 90 95
aaa tgc ctg gcc tgc atc aaa ctg ggc tct gag gac aaa gtt ctg ggc 336
Lys Cys Leu Ala Cys Ile Lys Leu Gly Ser Glu Asp Lys Val Leu Gly
100 105 110
cgg ttg gtc cac tgc ccc ata gag acc caa gtt ctg cgg ttt tgg gca 384
Arg Leu Val His Cys Pro Ile Glu Thr Gln Val Leu Arg Phe Trp Ala
115 120 125
ctg gca gga ggc tga 399
Leu Ala Gly Gly
130
<210>10
<211>132
<212>PRT
<213> Intelligent people
<400>10
Met Arg Arg Leu Leu Ile Pro Leu Ala Leu Trp Leu Gly Ala Val Gly
1 5 10 15
Val Gly Val Ala Glu Leu Thr Glu Ala Gln Arg Arg Gly Leu Gln Val
20 25 30
Ala Leu Glu Glu Phe His Lys His Pro Pro Val Gln Trp Ala Phe Gln
35 40 45
Glu Thr Ser Val Glu Ser Ala Val Asp Thr Pro Phe Pro Ala Gly Ile
50 55 60
Phe Val Arg Leu Glu Phe Lys Leu Gln Gln Thr Ser Cys Arg Lys Arg
65 70 75 80
Asp Trp Lys Lys Pro Glu Cys Lys Val Arg Pro Asn Gly Arg Lys Arg
85 90 95
Lys Cys Leu Ala Cys Ile Lys Leu Gly Ser Glu Asp Lys Val Leu Gly
100 105 110
Arg Leu Val His Cys Pro Ile Glu Thr Gln Val Leu Arg Phe Trp Ala
115 120 125
Leu Ala Gly Gly
130

Claims (15)

1. A method of making a population of cells comprising adherent stem cells, the method comprising:
obtaining a cell population in which the rate of adherent stem cells positive for KCNAB1 is 85% or more.
2. A cell population comprising adherent stem cells, wherein,
the cell population has a ratio of adherent stem cells positive for KCNAB1 of 85% or more.
3. The cell population of claim 2,
the relative expression amount of the SULT1E1 gene in the cell population with respect to the expression amount of the SDHA gene is 0.1 or more.
4. The cell population according to claim 2 or 3,
the relative expression level of the MN1 gene relative to the expression level of the SDHA gene in the cell population is 0.7 or more.
5. The cell population according to any one of claims 2 to 4,
the relative expression level of the RARES 2 gene in the cell population with respect to the expression level of the SDHA gene is 0.4 or less.
6. The cell population according to any one of claims 2 to 5,
the adherent stem cells are from a fetal appendage.
7. A pharmaceutical composition comprising the population of cells of any one of claims 2-6, and a pharmaceutically acceptable vehicle.
8. A pharmaceutical composition comprising a population of cells according to any one of claims 2 to 6, and other administrable cells.
9. The pharmaceutical composition according to claim 7 or 8, wherein,
1 dose of 10 for adherent stem cells in humans12Less than one/kg body weight.
10. The pharmaceutical composition according to any one of claims 7 to 9, wherein,
the pharmaceutical composition is an injection preparation.
11. The pharmaceutical composition according to any one of claims 7 to 9, wherein,
the pharmaceutical composition is a preparation for transplantation of a cell pellet or sheet-like structure.
12. The pharmaceutical composition according to any one of claims 7 to 11, which is selected from immune-related diseases, ischemic diseases, lower limb ischemia, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, nervous system diseases, graft-versus-host disease, inflammatory bowel disease, crohn's disease, ulcerative colitis, radiation enteritis, systemic lupus erythematosus, connective tissue diseases, stroke, cerebral infarction, intracerebral hematoma, cerebral palsy, liver cirrhosis, atopic dermatitis, multiple sclerosis, psoriasis, epidermolysis bullosa, diabetes, mycosis fungoides, scleroderma, diseases caused by degeneration and/or inflammation of connective tissues such as cartilage, articular cartilage defects, meniscus injury, osteochondritis dissecans, aseptic necrosis, knee osteoarthritis, inflammatory arthritis, rheumatoid arthritis, ocular diseases, angiogenesis-related diseases, and the like, A therapeutic agent for diseases in ischemic heart disease, coronary heart disease, myocardial infarction, angina pectoris, heart failure, cardiomyopathy, valvular heart disease, trauma, epithelial injury, fibrosis, pulmonary disease, and cancer.
13. A method of monitoring adherent stem cells for karyotypic abnormalities, the method comprising:
the method comprises the steps of measuring the ratio of adherent stem cells positive to KCNAB1 in a cell population containing the adherent stem cells, and monitoring the karyotype abnormality of the adherent stem cells by using the ratio of the adherent stem cells positive to KCNAB1 in the cell population of more than 85% as an index.
14. A method of evaluating a donor and/or a biological sample collected from a donor, the method comprising:
a cell population containing adherent stem cells is collected from a donor, the ratio of adherent stem cells positive for KCNAB1 is measured, and the ratio of adherent stem cells positive for KCNAB1 in the cell population is evaluated as an index of 85% or more.
15. A method of determining and/or predicting optimal enzyme treatment conditions for a biological sample, the method comprising:
the percentage of adherent stem cells positive for KCNAB1 in a cell population obtained by subjecting a biological sample collected from a donor to an enzyme treatment was measured, and the evaluation was performed using as an index the percentage of adherent stem cells positive for KCNAB1 in the cell population of 85% or more.
CN201880084442.5A 2017-12-28 2018-12-28 Cell population comprising adherent stem cells, method for producing same, and pharmaceutical composition Pending CN111527198A (en)

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